This event will highlight and discuss recent advances in strategies for controlling stem cell fate and reprogramming (including new insights into the molecular basis of pluripotency and differentiation)

together with the progress towards therapeutic and bioprocessing.

This event has CPD accreditation

This abstract book will be finalised two weeks before the event www.regonline.co.uk/stem2015

Table of Contents

Day 1: ..............................................................................................................................................................................................5

Invited Speakers Abstracts .................................................................................................................................................5

MicroRNA Activity and Adult Stem Cell Differentiation: Utilising the Epigenome ......................................................................5

Applications of in vitro assays to measure cellular response in 3D cell cultures ........................................................................5

3D-in vitro models - where are we and where do we go from here? ..........................................................................................5

Human stem cells as tools to model neural damage and cell death pathways ...........................................................................5

CONDITIONED MEDIUM OF HUMAN MESENCHYMAL STEM CELLS ENHANCE KERATINOCYTE MIGRATION INTO THE INJURY SITE

......................................................................................................................................................................................................5

NOVEL 3D BIO-RESPONSIVE MESENCHYMAL STEM CELL NICHE MODEL ....................................................................................6

Poster Presentation Abstracts ...........................................................................................................................................7

EXTRACELLULAR MATRIX AND GROWTH FACTORS REGULATE MYOGENESIS .............................................................................7

A SORTING STRATEGY FOR BOVINE MAMMARY PROGENITORS .................................................................................................7

ANALYSIS OF RELATION OF BREAST CANCER STEM-LIKE CELL MARKERS TO OTHER PROGNOSTIC FACTORS OF THE DISEASE .8

ARE MUSCLE-DERIVED CELLS AND MESENCHYMAL STEM CELLS GOOD CANDIDATES FOR CO-TRANSPLANTATION?- AN IN VITRO

RECIPROCAL INTERACTIONS ........................................................................................................................................................8

MCF-7 STEM-LIKE CELLS PRESENT INCREASED RESISTANCE TO IONIZING RADIATION IN VITRO, IN COMPARISON TO MCF-7

PARENTAL CELLS ..........................................................................................................................................................................9

A MEDICAL HYPOTHESIS: MITOCHONDRIA MALFUNCTIONS AS MEDIATORS OF STEM CELLS’ RELATED CARCINOGENESIS

SUPPORT THE HIGHLY CONSERVED PROFILE OF CARCINOGENESIS ......................................................................................... 10

MESENCHYMAL STEM CELLS PRESENTED DIFFERENCE IN BEHAVIOUR BETWEEN 3D SCAFFOLDS AND IN 2D CULTURE ........ 10

Day 2: ........................................................................................................................................................................................... 11

Invited Speakers Abstracts .............................................................................................................................................. 11

The impact of culture on properties of pluripotent stem cells ................................................................................................. 11

Direct Conversion of Pluripotent Human Embryonic Stem Cells into Functional Human Neuronal or Cardiomyocyte Cell Therapy

Derivatives for Regenerative Medicine ..................................................................................................................................... 11

Pluripotent stem cell models to understand human heart disease ......................................................................................... 11

Functional characterization of PCL embedding collagen composite scaffolds for mechanically-induced differentiation of hMSCs.

................................................................................................................................................................................................... 11

Cell therapy bioprocesses to support organ transplants .......................................................................................................... 11

Stem Cell Bioprocessing: The Role of Metabolism ................................................................................................................... 11

Clinical grade mesenchymal stromal/stem cells from umbilical cord Wharton's Jelly - should body mass index be a criterion for

donation? .................................................................................................................................................................................. 12

Challenges of industrializing stem cell production - a manufacturing perspective .................................................................. 12

Oral Presentation Abstracts ............................................................................................................................................ 12

ARE MESENCHYMAL STEM CELLS SUITABLE FOR LUNG THERAPY? .......................................................................................... 12

HIGHER CARDIOGENIC POTENTIAL OF INDUCED PLURIPOTENT STEM CELLS DERIVED FROM ATRIAL MESENCHYMAL STROMAL

CELLS COMPARED TO SYNGENEIC SKIN CELLS .......................................................................................................................... 13

UPSTREAM AND DOWNSTREAM SOLUTIONS FOR MSCs ANIMAL ORIGIN-FREE PROCESSING ................................................ 13

MESENCHYMAL STEM CELLS FROM UMBILICAL CORD BLOOD DIFFERENTIATE INTO KERATINOCYTES: USING AN ENZYME

ACTIVITY APPROACH TO FOLLOW CELL DIFFERENTIATION ...................................................................................................... 13

Poster Presentation Abstracts ........................................................................................................................................ 14

FUNCTIONAL 3D HUMAN HEPATOCYTE SPHEROIDS MADE BY CO-CULTURING DIFFERENTIATED HEPATOCYTES FORM HUMAN

ADIPOSE-DERIVED STEM CELLS AND UNDIFFERENTIATED HUMAN ADIPOSE-DERIVED STEM CELLS ...................................... 14

EX-VIVO GENERATION OF FULL TERM HUMAN AMNIOTIC FLUID MESENCHYMAL STEM CELLS IN XENO- AND SERUM-FREE

CONDITION ................................................................................................................................................................................ 14

ADIPOSE TISSUE-DERIVED MESENCHYMAL STEM CELLS IN COMBINATION WITH CYCLOSPORINE A MODULATE MACROPHAGE

POLARISATION AND SUPRESS INFLAMMATORY REACTION AFTER SKIN TRANSPLANTATION ................................................. 15

INTERFERON-γ-TREATED BONE MARROW MESENCHYMAL STEM CELLS INHIBIT PRODUCTION OF IL-10 BY B CELLS VIA

UPREGULATION OF COX-2 ........................................................................................................................................................ 15

IN VIVO IMAGING SYSTEM FOR ANALYSIS OF LARGE ANIMAL DERIVED EXPLANTS AS A METHOD FOR EVALUATION OF CELL

TRANSPLANTATION PROCEDURE EFFECTS ............................................................................................................................... 16

FATTY ACID AMIDE HYDROLASE INHIBITORS INDUCE MESENCHYMAL STEM CELL MIGRATION AND DIFFERENTIATION INTO THE

OSTEOBLASTIC LINEAGE ............................................................................................................................................................ 16

Day 3: ........................................................................................................................................................................................... 17

Invited Speakers Abstracts .............................................................................................................................................. 17

Identifïcation of novel factor enhacing homing of mesenchyma (stromal) stem cells to the injured tissues.......................... 17

Innovating Technologies to Enable Regenerative Medicine Manufacture ............................................................................... 17

Molecular toxicity of nanoparticles using mouse bone marrow mesenchymal stem cells ...................................................... 17

Driving Regenerative Medicine Biomanufacturing Scalability through Technology Repurposing and Academic:Industry

Partnerships .............................................................................................................................................................................. 18

Using pluripotent stem cells to treat age-related macular degeneration ............................................................................... 18

Oral Presentation Abstracts ............................................................................................................................................ 18

ANALYSIS OF RELEVANT OUTCOME PARAMETRES IN PATIENTS WITH ISCHEMIC CARDIOMYOPATHY THAT PREDICT RESPONSE TO

CD 133+ REGENERATIVE THERAPY ............................................................................................................................................ 18

Poster Presentation Abstracts ........................................................................................................................................ 19

CANCER STEM CELL MARKER IN CIRCULATING TUMOR CELLS OF HUMAN COLORECTAL CANCER: EXPRESSION OF CD44VARIANT

EXON9 IS STRONGLY CORRELATED TO TREATMENT REFRACTORINESS, RECURRENCE, AND PROGNOSIS .............................. 19

AN IN VITRO SYSTEM AS A TOOL ALLOWING TO EVALUATE HEMATOPOIETIC STEM CELLS LYMPHOPOIETIC CAPABILITY IN HIV

INFECTED INDIVIDUALS. ............................................................................................................................................................ 19

TARGETED NANOPARTICULATE DELIVERY OF BIOLOGIC PAYLOADS TO NEURAL (STEM) CELLS .............................................. 20

THE COMPARATIVE ANALYSIS OF ACUTE MACROPHAGES INFILTRATION AFTER TRANSURETHRAL TRANSPLANTATION OF

DIFFERENT CELL TYPES .............................................................................................................................................................. 20

Day 1:

Invited Speakers Abstracts MicroRNA Activity and Adult Stem Cell Differentiation: Utilising the Epigenome Assistant Professor Christopher J. Payne, PhD, Human Molecular Genetics Program, Ann & Robert H. Lurie Children's Hospital of Chicago Research Center, Departments of Pediatrics and Obstetrics and Gynecology, Northwestern University Feinberg School of Medicine, Chicago, IL, US Epigenetic processes influence stem cell differentiation. The combination of non-coding RNAs and histone modifications regulates lineage-specific gene expression and cell fate decisions. MicroRNAs and methylated lysine residues on histone tails play a particularly important role in many differentiation events. Characterisation of stem cells from the mammalian testis and umbilical cord has identified microRNA146a as a key epigenetic regulator. Its activity impacts Polycomb group histone methyltransferases and Jumonji domain containing histone demethylases, affecting the commitment of stem cells to differentiate. Modulation of microRNA146a in adult stem cells could provide new methods to optimize differentiation protocols for clinical applications and future therapies. Applications of in vitro assays to measure cellular response in 3D cell cultures Dr Craig Malcolm, Strategic Collaborations Manager, Cell-Based Assays & Emerging Technologies, Promega, Southampton, UK Three-dimensional cell culture provides a more physiologically relevant model compared to the traditional 2D approach. The 3D systems are able to more closely mimic the complex and heterogeneous in vivo microenvironment in terms of both intercellular structure and the presence of different cell types interspersed with extracellular matrix. The increasing popularity and availability of in vitro 3D models have created a demand for reliable cell-based assays capable of measuring different aspects of cellular physiology in a 3D environment. Promega has developed a range of cell-based assays which are compatible with 3D cell culture models. The range includes bioluminescent detection of ATP as a measure of cell viability, a caspase assay for detecting apoptosis and cell stress reporter assays to detect mechanisms leading to cytotoxicity. Furthermore our new viability and cell death assays can be performed in real-time. The non-lytic nature of our real-time assays allows the 3D cell cultures and microtissues to be used for further downstream applications and enables multiplexing with a variety of assay chemistries. 3D-in vitro models - where are we and where do we go from here? Dr Arno Gutleb,PhD, ERT, Luxembourg Institute of Science and Technology (LIST), Environmental Research and Innovation (ERIN) Department, Luxembourg Complex 3D in vitro coculture systems are valuable tools to study biological processes in a more realistic way than in the classic monocell and monolayer systems. Such 3D systems allow for example direct cell-cell interaction, cell migration, etc. that may affect cellular responses. In combination with modern systems biology approaches 3D in vitro co-culture systems are the next step to further replace in vivo experiments in drug development and toxicity studies. Complex 3D in vitro coculture systems are valuable tools to study biological processes in a more realistic way than in the classic monocell and monolayer systems. Such 3D systems allow for example direct cell-cell interaction, cell migration, etc. that may affect cellular responses. In combination with modern systems biology approaches 3D in vitro co-culture systems are the next step to further replace in vivo experiments in drug development and toxicity studies.Oral Presentation Abstracts Human stem cells as tools to model neural damage and cell death pathways Patrizia Ferretti, UCLInstitute of Child Health, London, United Kingdom Development of human in vitro 2D and 3D models relevant to modelling the human nervous system and their use for characterizing neural cell response to damage and novel putative neuroprotective agents will be discussed.

CONDITIONED MEDIUM OF HUMAN MESENCHYMAL STEM CELLS ENHANCE KERATINOCYTE MIGRATION INTO THE INJURY SITE Moyassar B H Al-Shaibani*1, Xiao N Wang1, Penny E Lovat1, Anne M Dickinson1 *1 Newcastle University, Institute of Cellular Medicine, Newcastle upon Tyne, United Kingdom Introduction: It has been reported that keratinocyte migration into the injury site is important for wound healing. The mechanism responsible for enhanced wound healing in the skin includes restorative paracrine effects of mesenchymal stem cells (MSCs) synthesised growth factors. Therefore, this research was

conducted to study the effect of MSC-conditioned medium (MSC-CM) in promoting keratinocyte migration into the injury site. Methods: Human MSCs were derived from hip joints, expanded in vitro and characterised morphologically, phenotypically and differentially. Conditioned medium was generated from MSC cultures in serum free DMEM medium supplemented antibiotics when cultures were approximately 80% confluent, then collected after 24 h (CM24), 48 h (CM48 and 72 h (CM72) and all cellular debris were removed by filtration (0.2 μ filter). The effect of this MSC-CM on migration of a human keratinocyte cell line (HaCat) was evaluated using a 2D scratch assay. Results: The generated MSCs met the criteria as assigned by the International Society for Cell Therapy (ISCT) by being fibroblast like cells with plastic adherence and capable of proliferating under in vitro conditions. Also, all cells demonstrated the ability to differentiate into three lineages; osteoblast, adipocyte and chondrocyte. Phenotypically, over 95% of the cells expressed CD73, CD90 and CD105 (P=0.0001), while being negative for the expression of CD14, CD19, CD34, CD45 and HLA-DR (P=0.025). Additionally, In 2D scratch assays, MSC-CM from different passages significantly enhanced wound closure of HaCat keratinocytes. For example, CM72 was the most effective at accelerating wound closure after 17 hr of treatment (P=0.0009). In addition, both CM48 and CM24 significantly minimised scratch areas after 17 hr of treatment (P=0.0011). Conclusion: Keratinocyte migration is a main mechanism which could be utilised to promote wound healing. The scratch assay could be applied in vitro to monitor keratinocyte mobility after treatment with test substances such as growth factors. These results suggest that MSC-CM contain effector molecules required for cell migration in the wounded region. A possible explanation for promoting keratinocyte mogration into the scratched area is that MSC-CM contain cytokines which cause accelerated cell migration such as nerve growth factor (NGF), IL-6, IL-8 and stromal derived factor-1 (SDF-1). Additionally, MSC-CM might contain other cytokines which can promote both keratinocyte migration and proliferation such as hepatocyte growth factor (HGF), platelet derived growth factor (PDGF) and granulocyte-macrophage colony stimulating factor (GM-CSF).

NOVEL 3D BIO-RESPONSIVE MESENCHYMAL STEM CELL NICHE MODEL E. E. L. Lewis, H. Wheadon, M. J. Dalby and C. C. Berry Centre for Cell Engineering, University of Glasgow, Glasgow, G12 8QQ, UK Mesenchymal stem cells (MSCs) are able to aid the regeneration of damaged tissue and organs through their ability to self-renew and differentiate into other cell types (bone, cartilage and fat).1 These cells reside within a special microenvironment within the bone marrow known as the niche, which regulates MSC fate and preserves stem cell number.2 Local injury is known to stimulate MSCs within the niche, causing cell migration and homing towards damaged tissue to aid the wound healing process.3 Exploration of the niche milieu will lead to greater understanding of the mechanisms regulating stem cell migration, homing and differentiation, with the view to allowing artificial manipulation for regenerative medicine. The study presented here offers a novel, regeneration-responsive, MSC niche model. Briefly, MSCs were loaded with magnetic nanoparticles (mNPs) and subjected to an external magnetic field to form multicellular spheroids. These spheroids remained quiescent with high viability, and exhibited high nestin and STRO-1 expression over time compared to traditional 2D cultured MSCs. The MSC spheroids were subsequently sited within a collagen gel, mimicking the in vivo bone marrow environment. The model was subjected to a series of bone (osteoblast) and connective tissue (fibroblast) wound healing assays, using a simple monolayer scratch cultured beneath the collagen gels. In the presence of a scratch, MSCs directionally migrated out of the niche and homed towards the site of injury becoming fully integrated into the wound. In addition, the MSCs were capable of differentiation into the appropriate cell type, with high levels of RUNX2 and osteopontin expression, at days 3 and 14 respectively, noted in response to the osteoblast wound assays, but not with fibroblast assays. Consequently, a multiplex assay was performed, which noted elevated levels of cytokine IL-6 secretion in both wound models. IL-6 was then added to the collagen matrix of the cultured MSC spheroids at identical concentrations, resulting in MSC migration out of the spheroid following 3 hours incubation onwards. In conclusion, we have developed an entirely novel physiologically relevant MSC niche model, whereby the MSCs are regeneration-responsive, migrating towards IL-6 signals from injured cells and differentiating appropriately. References: 1. H. Kagami et al., The International Journal of Biochemistry & Cell Biology, 2011, v43, p286. 2. I. Roeder et al., Blood Cells, Molecules, and Diseases, 2011, v46, p308. 3. B. Joddar and Y. Ito, Journal of Biotechnology, 2013, 168, p 218.

Poster Presentation Abstracts Poster abstracts will be finalised weeks before the event EXTRACELLULAR MATRIX AND GROWTH FACTORS REGULATE MYOGENESIS N. Walker and C. Niesler Department of Biochemistry, School of Life Sciences, University of KwaZulu-Natal, Private Bag X01, Scottsville 3209, South Africa 2015 Upon muscle injury, satellite cells are activated to myoblasts that subsequently proliferate, migrate and differentiate into myotubes in order to facilitate muscle repair. Extracellular matrix (ECM) factors in the satellite cell niche (primarily laminin and collagen IV) and wound (fibrotic matrix components such as collagen I and decorin) regulate aspects of myogenesis. At the molecular level, this process is controlled by a range of transcription factors including Pax7, MyoD and myogenin. Hepatocyte growth factor (HGF) initiates activation of quiescent satellite cells in the niche. It is also known to regulate differentiation; however its effect on this process in the presence of niche matrix factors is unclear. By differentiating C2C12 murine myoblasts in the presence of these ECM and growth factors we were able to assess their effect on the percentage Pax7+ and MyoD+ cells, as well as myotube fusion. In conclusion, ECM factors are able to modulate the effect of growth factors on myogenesis. Our results provide new insight into potential mechanisms by which this is achieved. A SORTING STRATEGY FOR BOVINE MAMMARY PROGENITORS S. Capano1, E. Martignani1, S. Miretti1, M. Baratta1,2 1 Department of Veterinary Science, University of Turin, Largo Braccini, 2, 10095 Grugliasco (TO), Italy 2corresponding author – email: mario.baratta@unito.it. The mammary gland is a bilayered system organized into a series of branching ducts that terminate in secretory alveoli. Their epithelium is composed of an inner layer of cytokeratin(CK)18+ luminal cells, which secrete milk, and an outer layer of contractile CK14+ myoepithelial cells, which contribute to milk ejection during lactation. The existence of mammary renewable stem cells (MaSCs) has been deduced from the profound regenerative ability of the mammary epithelium. It undergoes different morphogenetic changes during puberty and during the subsequent pregnancies. The knowledge of MaSCs properties and functions should represent an interesting opportunity in the field of animal production as the appropriate regulation of these cells should benefit milk yeld. The aim of our study is to characterize the MaSCs population of bovine species and set up a sorting strategy to purify different primitive populations. The Colony-Forming Cell (CFC) assay performed on bovine mammary samples obtained from virgin, two years old cows revealed the presence in the mammary gland of both myoepithelial and luminal-restricted progenitor cells. In order to select and isolate the different types of progenitors we have performed a flow cytometric analysis of cells dissociated from bovine mammary tissue. Cells were then stained with antibodies directed to CD49f, whose expression at high levels is associated with progenitor and stem cell activity, and p-cadherin, that is normally expressed in the myoepithelial/basal layer of the mammary epithelium. After depletion of hematopoietic cells (using an anti-CD45 antibody) we identified four different subpopulations. They were characterized by different levels of CD49f/P-cad expression: CD49f/P-cad- (15,6%), CD49f++/P-cad- (25,4%), CD49f+/P-cad+ (27,1%), CD49f+/P-cad++ (10,2%). After sorting, the CFC assay performed with the cells of the different subpopulations showed that CD49f++/P-cad- population was able to generate colonies with an higher frequency compared to the others, revealing an enrichment in stem cells. In particular, 35,7% of colonies showed a myoepithelial phenotype and 64,3% a luminal one. In order to analyze whether these cells also had an in vivo tissue regenerative activity, the four subpopulation were implanted in collagen gels under the kidney capsule of NOD/SCID immunodeficient mice in numbers proportional to their frequency in the CD45- population. Immunohistochemical analysis of gels recovered 4 weeks later revealed that only CD49f++/P-cad- subpopulation was able to regenerate in vivo mammary alveolar structures characterized by a lumen surrounded by two layers of cells: an inner K18+ layer and an outer K14+ layer. The CFC assay performed with single cell suspension obtained from recovered gels showed a higher colony-forming ability of CD49f++/P-cad- population compared to the others. Therefore the structures produced in these gels contained clonogenic progenitors, that were able to form the same types of colonies observed from freshly isolated cells (36,2% of myoepithelial colonies and 63,8% of luminal ones). By using a novel sorting strategy we show that the mammary myoepithelial cell layer can be divided in different subpopulations. Each one has a different progenitor content and only in one fraction (CD49f++/P-Cad-) we were able to detect a subset of adult stem cells able to regenerate a mammary epithelium. This sorting strategy might prove useful for understanding the stem cells population dynamics during the different phases that the mammary gland undergoes. Besides, given the similar organization of the mammary tissue in the bovine and the human species, the cow might represent a much better animal model than the mouse.

ANALYSIS OF RELATION OF BREAST CANCER STEM-LIKE CELL MARKERS TO OTHER PROGNOSTIC FACTORS OF THE DISEASE Matrona Frangou1, Emmanouel Plemmenos1, Despoina Karandrea1, Effrossyni Boutou2 and Dimitris Vlachodimitropoulos3

1. Areteion University Hospital, Medical School, Athens University 2. Dept of Biochemistry & Molecular Biology, Faculty of Biology, School of Sciences, Athens University,

Panepistimiopolis Ilissia, 15784, Athens, Greece, email:<eboutou@biol.uoa.gr> 3. Lab of Forensic Medicine & Toxicology, Medical School, Athens University, Greece

INTRODUCTION: The cancer stem cell hypothesis claims that tumors contain a subpopulation of cells with the potential of tumor regeneration and regrowth, being at the same time more resistant to chemo- and radio-therapies. In breast cancer these stem-like cells seem to be CD44+ ,CD24-/low and in some cases CD133 antigen is also expressed. These cell surface antigens are characterized as markers of cancer stem-like cells in breast tumors. According to this hypothesis we sought to evaluate the correlation between the immunohistochemical expression of these antigens and the clinicopathological features that may have prognostic significance for breast cancer patients. METHODS: 39 tissue samples from breast cancer tumors, embedded in paraffin, were collected and prepared as 4μ thick sections for immunohistochemistry on an Automatic Ventana System. Monoclonal antibodies against CD44 (NovoCastra), CD24 and CD133 (BIOCARE) were used. In all cases the grading, hormonal receptor status, c-erbB-2 expression, Ki-67 index and p53 gene expression were previously analyzed. Lymph node status was known for only a subset of these cases. RESULTS: Quite interestingly, there was no relation found between the expression of these markers and the differentiation of the tumor cells as well as with lymph node status together with p53 expression. In relation to the estrogen receptor status we found that CD24 was expressed in the 80% of the negative cases versus 25% of the positive cases (p=0,02), while CD44 and CD133 had no difference in their percentages of expression. CD 44 was expressed in the 39,2% of the negative cases of cerbB-2 versus 72,7% of positive cases.(p=0,060) .The Ki-67 index showed statistical significant correlation with the CD24 expression (68,1%for >15%and 35,2%<15%). DISCUSSION: According to our results it seems that CD44 expression was more commonly found co-expressed in cerbB-2positive cases possibly rendering these cancer cells more tumorigenic. In addition, quite unexpectedly, it seems that if a population of breast cancer tumor cells express in high degree CD24, this tumor tends to have poorer prognosis as it is related with negative ER status and high Ki-67 index. It is considered that ER -negative stem cells may represent the more primitive mammary stem cells. While more work is definitely required, we believe that this information can be useful both to the oncologist in order to plan a more effective treatment as well as to set up a basis for further expression profiling studies to this type of cells. ARE MUSCLE-DERIVED CELLS AND MESENCHYMAL STEM CELLS GOOD CANDIDATES FOR CO-TRANSPLANTATION?- AN IN VITRO RECIPROCAL INTERACTIONS A. Kulesza1, I. Szczepańska1, W. Zarychta1, M. Butrym1, B.. Pajak2, L. Paczek1, A. Burdzinska1

1Department of Immunology, Transplantology and Internal Diseases, Medical University of Warsaw, Warsaw, Poland 2Electron Microscopy Platform, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland presenting author: Agnieszka Kulesza, Dept. Immunology, Transplant Medicine and Internal Diseases. Medical University of Warsaw, ul. Nowogrodzka 59, 02-006 Warsaw, Poland, corresponding author: Anna Burdzinska, Dept. Immunology, Transplant Medicine and Internal Diseases. Medical University of Warsaw, ul. Nowogrodzka 59, 02-006 Warsaw, Poland, e-mail: anna.burdzinska@wum.edu.pl Introduction. Urinary incontinence (UI) is a common socio-medical problem that is most prevalent among middle-aged and elder women. The new approach in the UI treatment is to strengthen the impaired urethral sphincter by cellular therapy. The best cell population candidate for this application has not been determined yet. It is obvious to propose muscle derived cells (MDCs) as population with established high myogenic potential, however recently more attention is focused on mesenchymal stem cells (MSCs), due to their immunomodulatory properties. An interesting alternative is the simultaneous transplantation of both mentioned populations, potentially allowing a beneficial interplay between them.

The objective of the presented study was to evaluate the reciprocal interaction between MDCs and MSCs populations in terms of: 1) myogenic differentiation, 2) cell proliferation, and 3) cell migration. Methods and results. Caprine bone marrow derived MSCs and skeletal muscle derived cells were isolated and characterized by expression of surface antigens, immunocytochemistry and differentiation assay. To examine the influence of MDCs on MSCs myogenic differentiation the direct and indirect co-culture was performed. The direct co-culture of unlabeled MDCs and GFP+ MSCs demonstrated that MSCs are able to contribute myotubes formation. An indirect co-culture on MDCs placed on 1 µm pore Ø inserts followed by immunostaining of MSCs (cultured in basal compartment) against desmin revealed no expression of this myogenic marker in treated MSCs. To confirm the result, MSCs were treated with MDCs conditioned medium and desmin expression was assessed by Western blot. Again, no desmin expression was detected in MSCs. The opposite interaction was also tested: the influence of MSCs soluble factors on myogenic differentiation of MDCs was assessed by calculation of fusion index after culture in close vicinity with either MSCs or MDCs placed on 1 µm pore inserts. The analysis revealed that MSCs indirect neighborhood had no significant effect on MDCs myogenic differentiation comparing to cells cultured in vicinity of MDCs. To evaluate the reciprocal interaction between studied cell types in term of proliferation activity, both populations were treated with MSCs or MDCs conditioned media and BrdU test was performed. The analysis revealed that MSCs proliferation rate did not differ depending on the population used for conditioning whereas the MDCs proliferated more efficiently when cultured in MDC-conditioned medium comparing to those cultured in medium collected from MSCs. The cells migration capability was assessed using fluorophore labeled cells placed on 8 µm pore inserts in the presence or absence of cells (MDCs or MSCs) in the basal compartment. The fluorescence derived from migrated cells was quantified using Cell Imaging Multi-Mode Reader. The analysis revealed that the presence of cells (regardless of cell type) in the basal compartment significantly increased the migration of both studied populations. In conclusion, obtained results demonstrate that caprine MSCs are able to contribute muscle formation under influence of muscle derived cells, but rather by fusion not by induction of myogenic pathway by soluble MDCs derived factors. Moreover, presented preliminary data suggest that co-transplantion would not affect the migration capability of both examined populations. MCF-7 STEM-LIKE CELLS PRESENT INCREASED RESISTANCE TO IONIZING RADIATION IN VITRO, IN COMPARISON TO MCF-7 PARENTAL CELLS VI Hatzi, M Karakosta, M Louka, E Boutou, K Barszczewska, CE Vorgias, D Vlachodimitropoulos, GE Pantelias, GI Terzoudi. Please note, that instead of Karakosta M., the presenting Author is Hatzi VI Postal address of presenting Author: Laboratory of Health Physics, Radiobiology & Cytogenetics, Institute of Nuclear & Radiological Sciences & Technology, Energy & Safety (I.N.RA.S.T.E.S.), National Center for Scientific Research "Demokritos", 60037 Aghia Paraskevi, Athens, Greece Corresponding author name / e-mail: Vasiliki I. Hatzi / vh@ipta.demokritos.gr In clinical radiation therapy, the failure to clinically eradicate all tumor cells is considered a treatment failure. As such, the interest is focused towards the characteristics of the small population of cancer cells that can survive and are capable of repopulating the tumor after subcurative therapy including chemotherapy and radiation. Since the survival of cancer cell populations after radiotherapy suggests differences in radioresistance between different cell populations within the same tumor, the estimation of radiosensitivity levels of those distinct populations would provide valuable information in tumor therapeutics. For this purpose, we analyzed the radiosensitivity of MCF-7 stem-like cells, isolated as MCF-7-sphere-forming cells, in comparison to conventional MCF-7 cells. Based on the chromosomal radiosensitivity estimation and in order to elucidate the underlying mechanisms we also performed cell-cycle analysis by Premature Chromosome Condensation (PCC) following calyculin-A treatment. In our experimental procedure, MCF-7 cell cultures were treated with selected media in order to produce spheres of stem-like cells, according to the literature. Prior to chromosomal radiosensitivity analysis, the cells were irradiated with 0.5 Gy and colcemid was added to collect the cells at the subsequent metaphase. Following the standard cytogenetic fixation process, the cells were spread on microscopic slides and subjected to analysis using optical microscope. For each experimental point, 60 metaphases were analyzed and the number of chromatid type aberrations as well as the chromosome number was scored. Mean values and standard deviations were calculated from three independent experiments. Cell cycle analysis was performed by the addition of calyculin-A for 45 minutes before the fixation process. The percentage of cells in S-phase, G2 and M-phase were scored in irradiated and non-irradiated cells. The results have shown a statistically significant difference at the level of radiosensitivity between MCF-7 parental and stem-like cells. The MCF-7 spheres deriving from stem-like cells have shown to be significantly more radioresistant when compared to MCF-7 cells grown in a monolayer, indicating that distinct cell subpopulations respond differently to ionizing radiation in terms of chromosomal damage. Moreover, calyculin-A mediated cell cycle analysis using PCC has shown that the different cell populations tested show differences in their cell-cycle profile. Considering that, to our knowledge, radiation data specific to stem/progenitor cells from

human primary breast cancers have not been reported, the application of the chromosomal radiosensitivity assay in breast tumor cell lines and biopsies might contribute to the development of new and highly targeted therapies with increased efficacy. A MEDICAL HYPOTHESIS: MITOCHONDRIA MALFUNCTIONS AS MEDIATORS OF STEM CELLS’ RELATED CARCINOGENESIS SUPPORT THE HIGHLY CONSERVED PROFILE OF CARCINOGENESIS VI Hatzi, GI Terzoudi, M Karakosta, E Boutou, M Louka, CE Vorgias, D Vlachodimitropoulos, GE Pantelias, V Makropoulos Address of presenting Author: Laboratory of Health Physics, Radiobiology & Cytogenetics, Institute of Nuclear & Radiological Sciences & Technology, Energy & Safety (I.N.RA.S.T.E.S.), National Center for Scientific Research "Demokritos", 60037 Aghia Paraskevi, Athens, Greece Corresponding author name / e-mail: Dr. Vasiliki I. Hatzi / vh@ipta.demokritos.gr Cancer development is an evolutionary process that has been highly conserved among centuries within organisms. Based on this, the interest in cancer research focuses on cells, organelles and genes that possess a genetic conservatism from yeasts to human. Towards this thought, mitochondria, the highly conserved and responsible for the cellular bioenergetic activity organelles, might play crucial role in carcinogenesis. Interestingly, tumors with low bioenergetic signature have worse prognosis and show a decreased expression of ATPase protein. Furthermore, according to the stem-cell theory of carcinogenesis, aggressive tumors are characterized by an increase number of malignant stem-like cell population and their resistance to chemotherapy has been found to be mitochondrially driven. The above considerations triggered us to hypothesize that mitochondrial bioenergetic processes in stem-like cancer cells play a crucial role in the highly conserved process of carcinogenesis (Medical Hypothesis, 2013, 80(1):70-4). Specifically, we support that mitochondrial and/or nuclear DNA alterations that control stem cells' ATP production drive stem cells to "immortalization" (Otto Warburg theory) that mediates cancer initiation and progression. Substantiation of our hypothesis requires evidence that: (1) alterations in mitochondria bioenergetic metabolites and enzymes encoded either from the mtDNA or the nuclear DNA are linked to human cancer and (2) mitochondrial functions are regulated by highly conserved genes involved in cancer-related cellular processes such as apoptosis, aging and autophagy. In the present work, we present an experimental approach on how this hypothesis can be tested and promising strategies in cancer therapeutics are also presented. In case the hypothesis of stem-cell bioenergetic malformations' related carcinogenesis proves to be correct, it would contribute to the development of new prognostic, diagnostic and even more effective therapeutic interventions against various types of cancer. MESENCHYMAL STEM CELLS PRESENTED DIFFERENCE IN BEHAVIOUR BETWEEN 3D SCAFFOLDS AND IN 2D CULTURE Saeed Kabrah1,2, Jenny May1, Craig Donaldson3 and Ruth Morse1 1. Centre for Research in Biosciences, University of the West of England 2. Department of laboratory medicine, Faculty of Applied Medical Sciences, Umm Al-Qura University, Makkah, Saudi Arabia. 3. Centre for Research in Translational Biomedicine, Plymouth University Developing a novel three-dimensional (3D) in vitro model of the bone marrow microenvironment using bone marrow mesenchymal stem cells (BM-MSCs) in commercial matrices involves a series of distinct processes. This starts with selecting an appropriate cell source and growth environment. Then choosing the appropriate scaffold that mimic the in vivo situation of the bone marrow. Primary cells isolated from healthy donors have low proliferation rate and lack homogeneity that can result in experimental variation. This study evaluated the immortalized stromal cell line HS-5 as an alternative cell source and evaluated their behaviour in the three different 3D cultures (Biomerix, Nanofiber and Alvetex). Cell morphology and proliferation in the 3D scaffolds were evaluated and compared to determine the difference between the normal BM-MSCs and cell line. In addition, Cell viability and CD markers expression were compared between the 2D and the 3D cultures in seven days. Also cellular size were assessed and 12 reference genes expression was compared between these cultures. The results indicate that there was no difference between the normal and cell line in morphology, they both presented fibroblastic cell structure in 2D culture while presenting rounded cells in the 3D culture. BM-MSCs were not able to penetrate into the nonofiber matrices; however, HS-5 cells were able to grow inside the nanofiber and has different structure to those cultivated into the other scaffolds. In addition, there were significant difference in cell number between the 3D cultures and the 2D. Moreover, there were differences in cell size between the techniques as well as cell viability. Some of the cells grown into 3D culture was found floating in the medium during the culture period and some cells adhered to the well forming a monolayer. In addition, the expression level of the 12 reference genes was different between the 2D and the 3D culture, but also they have different expression level between the 3D cultures. In conclusion, BM-MSCs behaviour is different in 2D compared to 3D cultures. Also the difference between 3D scaffolds lead to variation in cellular behaviour.

Day 2: Invited Speakers Abstracts The impact of culture on properties of pluripotent stem cells Dr Kirsten McEwen, MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College London, London, UK The choice of culture condition has a major impact on diverse properties of pluripotent stem cells (PSCs). We and others have found that transcription, epigenetic and metabolic phenotypes of mouse PSCs are altered by culture in traditional serum-containing conditions compared to 2i conditions. This highlights the flexible and dynamic nature of the pluripotent state. These findings have led us to investigate the role of variability as a feature of the pluripotent state and to decipher the mechanisms regulating transcriptional variability in PSCs. This will enhance future strategies directing PSC reprogramming and differentiation. Direct Conversion of Pluripotent Human Embryonic Stem Cells into Functional Human Neuronal or Cardiomyocyte Cell Therapy Derivatives for Regenerative Medicine Dr. Xuejun H Parsons, San Diego Regenerative Medicine Institute & Xcelthera Inc, San Diego, CA, United States Given the limited capacity of CNS and heart for self-repair, cell-based therapy represents a promising therapeutic approach closest to provide a cure. Recent advances have overcome some major obstacles in moving stem cell research from animals towards humans trials, including resolving minimal essential human requirements for derivation of clinically-suitable stable human ES cell lines and direct conversion of such pluripotent cells into a large supply of clinical-grade functional human neuronal or cardiomyocyte cell therapy products. Such breakthrough stem cell technologies have presented human ES cell therapy derivatives as a powerful pharmacologic agent of cellular entity for CNS and heart repair. Pluripotent stem cell models to understand human heart disease Dr Gabor Foldes MD PhD, National Heart and Lung Institute, Imperial College London, Imperial Centre for Experimental and Translational Medicine, Hammersmith Campus, London, UK The potential of stem cell-derived cardiomyocytes for disease modelling has been enhanced by the realisation that cardiomyocytes from human embryonic stem cells and induced pluripotent stem cells can be obtained also with disease-specific genotypes and phenotypes. These cells are suggested to have many of the properties of authentic cells, and their phenotypes provide validation that characteristics of the disease can be reproduced in vitro. This talk will present various examples how these cells can be used in disease modelling and drug screening. Functional characterization of PCL embedding collagen composite scaffolds for mechanically-induced differentiation of hMSCs. Marzia Brunelli Ph.D, Department of Mechanical Engineering, University of Sheffield Mechanobiology aims to control mesenchymal stem cells differentiation by applying mechanical stimuli.The use of 3D structures as support for cell growth and mechanically induced osteogenesis is still demanding for a standard structure able to provide control over the mechanical environment resulting from applied external forces. This study propose a rapid prototyped 3D polycaprolacton scaffolds embedding collagen gel responsible for providing a cell-niche able to enhance cell proliferation and for transmitting the mechanical stress to seeded cells. The outcomes demonstrate the suitability of PCL scaffolds to be used as substrate for mechanobiology investigation and its limitation to low strain and low frequency applications. Cell therapy bioprocesses to support organ transplants Dr Marianne Ellis, Department of Chemical Engineering, University of Bath, UK Organ transplants save lives but there are still areas for improvement in terms of immunosupporession and tissue preparation priot to implantation. This talk will present bioprocessing approaches currently being developed to address these challenges Stem Cell Bioprocessing: The Role of Metabolism Professor Athanasios Mantalaris, Chemical Engineering Department, Imperial college, London Stem cell bioprocessing lags behind the established bioprocess industries and practices in terms of control, standardisation, optimisation, and cost-effectiveness (productivity). Even though product quality (cells) is well-established, process parameters and requirements remain fundamentally empirical and their control rudimentary. Metabolism is at the heart of understanding stem cell biology and controlling the bioprocess to ensure product quality. It plays a pivotal role in defining whether a cell proliferates, differentiates, remains quiescent, or enters apoptosis. These insights promise to inform strategies for the directed

differentiation of stem cells and to offer the potential for further improvements to stem cell bioprocessing towards clinical applications. Clinical grade mesenchymal stromal/stem cells from umbilical cord Wharton's Jelly - should body mass index be a criterion for donation? Dr Dusko Ilic, MD PhD Reader in Stem Cell Science, King's College London The umbilical cord is routinely discarded at birth; however it contains within Wharton’s Jelly (WJ), foetal mesenchymal stromal/stem cells (MSC) that can be isolated and expanded in vitro with a minimal manipulation. These WJ MSC are used in clinical trials for treatment of variety of diseases. However, there is little or no attention paid to whether these stromal/stem cells isolated from infants exposed to an abnormal metabolic maternal environment behave differently. We investigated whether the maternal environment in obese women induces highly gene-specific epigenetic changes in WJ MSC rendering them unsuitable for cell based therapy.

Challenges of industrializing stem cell production - a manufacturing perspective

Dr Ricardo Baptista, PhD, Lead Scientist – Process Development, Cell Therapy Catapult, London, UK The talk intends to highlight some of the challenges faced when industrilazing stem cell production, and introduce the apporach of The Cell Therapy Catapult in addressing some of these challenges.

Oral Presentation Abstracts ARE MESENCHYMAL STEM CELLS SUITABLE FOR LUNG THERAPY? C. Rio; A. Jahn; A. Iglesias, T. de Francisco; O. Gigirey; J. A. Torrecilla; Á. Carvajal; J. Verdú; L. A. Ortiz; E. Sala-Llinas Institut d'Investigació Sanitària de Palma (IdISPa), Planta -1, Módulo F, Hospital Universitari Son Espases, Ctra. de Valldemossa, 79 07010 Palma Illes Balears, Spain – Telf. +34-871205000 ext 64521, e-mail: carlos.rio@ssib.es Introduction: Mesenchymal Stem Cells (MSC) have been shown to contribute to pulmonary repair and regeneration in lung injury models, and there are even a few ongoing human clinical trials using these adult stem cells. However, in some lung diseases (i.e. Chronic Obstructive Pulmonary Disease [COPD] and Idiopathic Pulmonary Fibrosis [IPF]) MSC can be involved in their pathogenesis. We hypothesize that MSC, both from bone marrow (BM-MSC) and adipose tissue (ADSC), from COPD and IPF patients may have an altered functional and regenerative capacity. Aims: To study the molecular and functional response of MSC (BM-MSC and ADSC), obtained from COPD and IPF patients, compared to those obtained from controls. We have evaluated: 1) the cellular response of MSC to VEGF, PDFG and tobacco; 2) the expression level of some of these growth factor receptors; 3) their differentiation capacity; and, 4) the ‘regenerative’ potential of the MSC and their conditioned media in vitro. Methods: MSC were isolated from bone marrow of sternum or from surrounding fat from subjects undergoing thoracic surgery. MSC functional response was monitored with the xCELLigence system (ACEA biosciences) measuring real time changes in cellular impedance. The amount of growth factor receptors was analyzed by RT-PCR. MSC differentiation and tyrosine phosphorylation was studied with LI-COR’s Odyssey system. Results: 1) There seems to be a gradient response to tobacco stimulation, being MSC-COPD cells more sensitive than cells from other origins; 2) MSC from diverse origins differentially respond to VEGF 121, VEGF 165, PDGF-AA and PDFG-BB; 3) PDGF-BB elicits the greatest response in tyrosine phosphorylation and there is a different response depending on the pathology of origin (COPD or IPF); 4) There is a diminished differentiation potential to adipocytes in ‘pathological’ MSC; 5) The ‘regenerative’ potential of conditioned media from MSC from COPD (MSC-COPD) or IPF patients (MSC-IPF) on alveolar epithelial cells seems to be lower than that of control cells. Conclusions: We have found some molecular and functional differences in MSC from patients with some lung pathologies. This anomalous behavior could play a role in the development and/or progression of the diseases. Cautious consideration should also be taken in the use of MSC in autologous cell therapy. Supported by FIS PI12-01152 and SEPAR 2011 and 2013.

HIGHER CARDIOGENIC POTENTIAL OF INDUCED PLURIPOTENT STEM CELLS DERIVED FROM ATRIAL MESENCHYMAL STROMAL CELLS COMPARED TO SYNGENEIC SKIN CELLS V. Meraviglia, J. Wen, L. Piacentini, G. Campostrini, C. Wang, M.C. Florio, V. Azzimato, L. Fassina, M. Langes, J. Wong, M. Miragoli, C. Gaetano, G. Pompilio, A. Barbuti, D. DiFrancesco, D. Mascalzoni, P.P. Pramstaller, G.I. Colombo, H.S.V. Chen, A. Rossini. Center for Biomedicine, European Academy Bozen/Bolzano (EURAC) Via Galvani 31, 39100 Bolzano, Italy e-mail: viviana.meraviglia@eurac.edu Mesenchymal stromal cells (MSCs) from atrial tissue retain specific cardiac differentiation abilities and they differentiate into cardiovascular cell types better than syngeneic cells of non-cardiac origin both in vitro and in vivo. Prior studies have demonstrated that the founder cell type could influence the molecular and developmental properties of induced pluripotent stem cells (iPSCs) at early passages after establishing their pluripotent state. Aim of the present work was to evaluate whether iPSCs obtained from cardiac versus skin mesenchymal stromal cells from the same individual retain a functional memory of their tissue of origin. We found that, at passages > 15, iPSCs derived from atrial MSCs (A-iPSCs) produced a significantly higher number of beating embryoid bodies than iPSCs from skin MSCs (S-iPSCs). Flow cytometry analysis revealed that embryoid bodies and their dissected beating areas from A-iPSCs exhibit more Troponin-T positive cells when compared to S-iPSCs. Importantly, cardiomyocytes derived from A-iPSCs (A-iPSC-CMs) displayed more mature characteristics with higher expression of cardiac markers (sarcomeric proteins, ionic channels, and myomiRs), more hyperpolarized diastolic potentials, and larger action potential amplitude than S-iPSC-CMs. Kinematics and dynamics evaluation showed that A-iPSC-CMs were characterized by significantly higher contractility compared to S-iPSC-CMs. In addition, different microRNA subsets were differentially modulated in atrial vs skin MSCs during the reprogramming process, potentially accounting for the higher cardiogenic potentials of iPSCs derived from atrial vs skin MSCs. In conclusion, the present work demonstrates the existence of a “functional” memory of the founder organ and, as a consequence, display better cardiac differentiation ability. UPSTREAM AND DOWNSTREAM SOLUTIONS FOR MSCs ANIMAL ORIGIN-FREE PROCESSING L. Savary; A. Schnitzler; M. Aysola; A. Verma; T. Lawson; S. Rigby; M. Pease; S. Punreddy; D. Kehoe, K. Philbrick; A. Dupont; Q. Feng; J. Murrell; M. Rook Merck Millipore, 39 Route Industrielle de la Hardt, 67120 Molsheim, France Corresponding Authors: - A. Schnitzler, EMD Millipore Corporation, 80 Ashby Rd, Bedford, MA 01730, USA. aletta.schnitzler@emdmillipore.com - D. Kehoe, EMD Millipore Corporation, 80 Ashby Rd, Bedford, MA 01730, USA. daniel.kehoe@emdmillipore.com Abstract: The long-term view of regenerative medicine therapies predicts an increased need for expansion solutions that ease scalability, utilize animal origin-free materials and are compatible with limited downstream processing steps. As more stem cell therapeutics progress through clinical testing, current in vitro culture in 2D vessels are proving cumbersome to scale. Moreover, the concurrent increased demands for serum from the recombinant protein and vaccines markets may result in a shortage of serum as clinical cell therapy programs are successful. In addition, high quality animal origin-free reagents and downstream processing devices support the future implementation of large scale manufacturing solutions that will be required following clinical success. The presentation will review solutions addressing animal origin-free expansion of MSCs within the context of different upstream process development steps (media supplementation, detachment enzyme and enzyme inhibitors) as well as scaling and downstream processing up to the 50L scale (cell separation from microcarriers and cell concentration) with good cell quality, high recovery and high viability. MESENCHYMAL STEM CELLS FROM UMBILICAL CORD BLOOD DIFFERENTIATE INTO KERATINOCYTES: USING AN ENZYME ACTIVITY APPROACH TO FOLLOW CELL DIFFERENTIATION JF dos Santos1,2, AS Fornaziero1, MS Araújo1, AS Sardella2, AK Azevedo-Martins2 and VA Nunes2 Department of 1Biochemistry of UNIFESP and 2School of Arts, Sciences and Humanities of USP, São Paulo, Brazil. Presenting author’s address: Avenida Arlindo Bettio, 1000 CEP 03828-000 Email address: vanunes@ib.usp.br Phone number: (+55)11 3091-8828 Human skin is composed by a multilayered epidermis formed by keratinocytes, accessory structures, dermis and a fat layer underlying the subcutaneous tissue. Keratinocytes constantly renew, however,

pathological conditions such as psoriasis can affect cell proliferation and differentiation. Several approaches have been proposed to study and treat psoriasis including stem cells (SC) therapy. In this context, human umbilical cord blood (UCB) has been considered an important source of SC and has been explored in both research investigation and regenerative medicine. The aim of this work is to evaluate the potential of differentiation of mesenchymal stem cells (MSC) from UCB into keratinocytes and to study molecular events involved in this process. Adherent SC from UCB samples were cultured in Dulbecco’s modified Eagle’s medium, low glucose and 15% fetal bovine serum until reached a spindle shaped appearance. Cells were characterized by the presence of SC markers (CD44, CD29, CD90 CD105,CD73 CD34 and CD31) by flow cytometry. To evaluate MSC properties, cells were subjected to adipogenic, chondrogenic and osteogenic differentiation. Differentiation of MSC into keratinocytes was induced by culturing cells in a medium for keratinocyte development supplemented with EGF and insulin. Cultures were evaluated on different days (1, 3, 7, 14 and 21) based on the activity of proteases such as kallikreins 6 and 7. Cell morphology was monitored in these time points. Data showed high activity of a protease in cell lysates of 14 and 21-day cultures upon the substrate Abz-GFSPFRSSRQ-EDDnp. This activity was blocked by EDTA, suggesting the presence of metalloprotease(s) in the differentiation process of MSC into keratinocytes. In combination with morphology and immunofluorescence analysis for the expression of cytokeratin 5 and p63, these results open the possibility to use enzymatic assays to access the differentiation process of SC and to study molecular events involved in skin biology. Supported by FAPESP and CNPq. Poster Presentation Abstracts Poster abstracts will be finalised weeks before the event FUNCTIONAL 3D HUMAN HEPATOCYTE SPHEROIDS MADE BY CO-CULTURING DIFFERENTIATED HEPATOCYTES FORM HUMAN ADIPOSE-DERIVED STEM CELLS AND UNDIFFERENTIATED HUMAN ADIPOSE-DERIVED STEM CELLS In-Su Parka,*, Phil-Sang Chunga,b, Jin Chul Ahna,c,d,* a Beckman Laser Institute Korea, Dankook University, 119 Dandae-ro, Cheonan, Chungnam 330-714, Korea b Department of Otolaryngology-Head and Neck Surgery, College of Medicine, Dankook University, 119 Dandae-ro, Cheonan, Chungnam 330-714, Korea c Department of Biomedical Science, Dankook University, Cheonan, Chungnam, 330-714, Korea d Biomedical Translational Research Institute, Dankook University, Cheonan, Chungnam, 330-714, Korea * Corresponding author. Fax: +82 41 559 7838. E-mail address: jcahn@dankook.ac.kr (J.C. Ahn), insu0223@gmail.com (I.S. Park). Acritical shortage of donor organs for treating end-stage organ failure highlights the urgent need for generating organs from stem cells. Despite many reports describing functional cell differentiation, no studies have succeeded in generating a three-dimensional vascularized organ such as liver. We have generated human hepatocyte spheroids with uniform size and shape by co-culturing 1:1 mixtures of differentiated hepatocytes form hASCs and undifferentiated hASCs in low cell binding surface. The hASCs in spheroids could compensate for the low viability and improve the functional maintenance of human hepatocytes (hHeps). Co-cultured spheroids aggregated and formed compact spheroidal shapes more rapidly, and with a significantly higher viability than mono-cultured spheroids. The liver-specific functions of co-cultured spheroids were greater, although they contained half the number of hepatocytes as mono-cultured spheroids. Albumin secretion by cocultured spheroids was higher on day 14, whereas urea secretion was similar, compared with mono-cultured spheroids. Spheroids serve as individual vascularization units, promoting the simultaneous development of new microvascular networks at different locations inside implanted tissue constructs. These co-cultured spheroids may be useful for creating artificial three dimensional hepatic tissue constructs and for cell therapy with limited numbers of human hepatocytes. EX-VIVO GENERATION OF FULL TERM HUMAN AMNIOTIC FLUID MESENCHYMAL STEM CELLS IN XENO- AND SERUM-FREE CONDITION K. Thilakavathy1,2, P. Ghoraishizadeh2, R. Ramasamy1,3, Z. Rejali2, B.C. Tan4, G. Singh5, R. Rosli1,2 and N. Nordin1,2

1Genetics and Regenerative Medicine Research Centre, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM SERDANG, Selangor, Malaysia 2Department of Obstetrics and Gynaecology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Selangor, Malaysia 3Immunology Unit, Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Selangor, Malaysia 4Britannia Women and Children Specialist Centre, Selangor, Malaysia 5Stempeutics Research Malaysia Sdn. Bhd, Technology Park Malaysia, Kuala Lumpur, Malaysia

Human amniotic fluid which is generally considered as clinical waste during delivery, has been found to be a rich source of stem cells. However, suitable culture media in culturing full term human amniotic fluid mesenchymal stem cells (hAFMSCs), particularly for therapeutic purposes, has not been fully established. Commonly, basal medium supplemented with animal serum, mostly foetal bovine serum, is frequently used. Unfortunately, this condition has been associated with the risk of transmission of animal pathogens and xenogeneic immuno reaction. This study aimed to explore the feasibility to isolate and propagate full term human amniotic fluid mesenchymal stem cells (hAFMSCs) obtained during caesarean section deliveries using xeno- and serum-free medium (MesenCultTM-XF). The mesenchymal stem cells were characterised according to standard methods. The generated hAFMSCs showed spindle-shaped fibroblast-like morphology with an average population doubling time of 36hrs. These cells expressed more than 90% of MSCs positive markers (CD44+, CD73+, CD90+, CD105+, CD166+) and less than 1% of negative markers (CD14-, CD19-, CD34-, CD45-and HLA-DR-). hAF-MSCs generated in this xeno- and serum-free medium were capable of differentiating into adipocytes, osteocytes and chondrocytes. Our findings highly suggest that xenogenic free hAF-MSCs were feasible to be generated from full-term amniotic fluid, a waste product during delivery, for safe therapeutic application and possible hAF-MSCs banking. ADIPOSE TISSUE-DERIVED MESENCHYMAL STEM CELLS IN COMBINATION WITH CYCLOSPORINE A MODULATE MACROPHAGE POLARISATION AND SUPRESS INFLAMMATORY REACTION AFTER SKIN TRANSPLANTATION M Hajkovaa,b,*, B Hermankovaa,b, E Javorkovaa,b, A Zajicovab , P Trosana,b, V Holana,b, M Krulova a,b,* aFaculty of Science, Charles University, Vinicna 7, Prague 2, 128 43, Czech Republic, bInstitute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic *Corresponding author: M Krulova: krulova@natur.cuni.cz, M Hajkova: michaela.hajkova@natur.cuni.cz Mesenchymal stem cells (MSCs) are an attractive source of multipotent cells with immunomodulatory, antiapoptotic and cytoprotective capabilities. They have been used in regenerative medicine and treatment of many inflammatory diseases. Although several studies have demonstrated that the administration of MSCs in combination with an immunosuppressive drug prolongs allograft survival in comparison with use of MSCs or the drug alone, little is known about the exact mechanism of such synergism. Our study was focused on analysis of the therapeutic effect of MSCs-seeded and Cyclosporine A-loaded electrospun nanofibers in a mouse model of allogeneic skin transplantation. We observed that the treatment with MSCs and CsA significantly decreased the amount of graft-infiltrating macrophages and the production of nitric oxide. These changes were accompanied with the considerable lower production of interferon-gamma as a cytokine priming classically activated macrophages. Simultaneously, the significant upregulation of CD206 expression and the increased production of interleukin-10 by graft infiltrating macrophages as a markers of alternatively activated macrophages were detected. In this study we found that the local application of MSC-seeded and CsA-loaded nanofiber scaffolds modulate the macrophages towards alternatively activated phenotype. This phenotype switching may contribute to tissue regeneration and suppression of the local inflammatory reaction. INTERFERON-γ-TREATED BONE MARROW MESENCHYMAL STEM CELLS INHIBIT PRODUCTION OF IL-10 BY B CELLS VIA UPREGULATION OF COX-2 B. Hermankova1,2, A. Zajicova1, E. Javorkova1,2, M. Hajkova1,2, M. Chudickova1,2, P. Trosan1,2, M. Krulova1,2 and V. Holan1,2

1 Institute of Experimental Medicine, Academy of Science of the Czech Republic, Videnska 1083, Prague 4, 142 20, Czech Republic 2 Faculty of Science, Charles University, Vinicna 7, Prague 2, 128 43, Czech Republic Corresponding author: Barbora Hermankova (bhermankova@gmail.com) Mesenchymal stem cells (MSCs) represent a heterogenous population of nonhematopoietic stem cells with multipotent differential potential. MSCs are a good candidate for regenerative medicine and treatment of various diseases. They are able to suppress immune response by a cell contact, production of soluble factors and by other mechanisms. In this study we analysed the effects of mouse bone marrow-derived MSCs on IL-10 production by lipopolysaccharide-activated B cells. The production of IL-10 was significantly enhanced in the presence of interferon-γ (IFN-γ). Untreated MSCs had no significant effect on IL-10 production by B cells. However, when B cells were cocultured with MSCs and IFN-γ together, the production of IL-10 was strongly suppressed. Moreover, a similar effect was observed when MSCs were preincubated with IFN-γ and then cocultured with activated B cells. On the other hand, the production of IL-10 was at normal level after cultivation IFN-γ-pretreated B cells with MSCs. A strong upregulation of indoleamine-2,3-dioxygenase (IDO), cyclooxygenase 2 (COX-2) and programmed cell death ligand-1 (PD-L1) was detected in MSCs treated with IFN-γ. To identify the molecule which is responsible for the suppression of IL-10 production, we used neutralization monoclonal antibody anti-PD-L1 or inhibitors of

IDO and COX-2. The suppression of IL-10 production was abrogated only in cultures with inhibitor of COX-2. The suppressor effect of COX-2 was verified by adding prostaglandin E2, a product of COX-2, to the cultures with activated B cells. Altogether, the results show that the mechanism of MSC mediated inhibition of IL-10 production by B cells involves COX-2 molecule and paracrine secretion of prostaglandin E2. IN VIVO IMAGING SYSTEM FOR ANALYSIS OF LARGE ANIMAL DERIVED EXPLANTS AS A METHOD FOR EVALUATION OF CELL TRANSPLANTATION PROCEDURE EFFECTS W. Zarychta1, R. Zagożdżon2, M. Butrym1, A. Kulesza1, K. Siewruk3, L. Paczek1, A. Burdzinska1 1Department of Immunology, Transplantology and Internal Diseases, Medical University of Warsaw, Warsaw, Poland 2Department of Immunology, Center of Biostructure Research, Medical University of Warsaw, Warsaw, Poland 3Department of Large Animal Diseases with Clinic, Faculty of Veterinary Medicine, Warsaw University of Life Sciences (SGGW), Warsaw, Poland presenting author: Weronika Zarychta, Dept. Immunology, Transplant Medicine and Internal Diseases. Medical University of Warsaw, ul. Nowogrodzka 59, 02-006 Warsaw, Poland, corresponding author: Anna Burdzinska, Dept. Immunology, Transplant Medicine and Internal Diseases. Medical University of Warsaw, ul. Nowogrodzka 59, 02-006 Warsaw, Poland, e-mail: anna.burdzinska@wum.edu.pl Introduction. Urinary incontinence (UI) is a serious socio-medical problem which affects about 10% of human population. Cell transplantation has been proposed as an alternative method for the UI treatment. Although many results in this field are promising, the recommended protocol for such a procedure has not been determined. Therefore further pre-clinical studies are needed, preferentially on large animal models in order to mimic transurethral injections performed in human patients. Tracking of cells after transplantation is difficult in large animals. We propose the use of In Vivo Imaging System (IVIS) to analyze urethral explants after cell injection. Aim of the study. The objective was to assess whether the IVIS can be used for analysis of large animal derived explants in order to evaluate the effects of cell transplantation procedure. Material and methods. In vitro study. Cells labeled with lipophilic cyanine dye DID (Ex= 650, Em=670 nm) and PKH26 (Ex=551 Em= 567 nm) placed in 96-well plate were visualized with IVIS. Semi- quantitative evaluation of fluorescence total radian efficiency (TRE) according to concentration of cell suspension and then signal to background ratio was calculated. Ex vivo study. Urethras were obtained from adult female goats (age >6) 28 days after cells (n=9) or PBS (n=3) injection. Samples were divided into 5-7 tissue thick cross-sections and analyzed with the use of IVIS. Automatic algorithm spectral unmixing followed or not by manual setup was used to separate specific dye signal from tissue autofluorescence. The results were verified by fluorescence microscopy of corresponding cross-sections. Results. TRE is directly proportional to concentration of cells labeled with both dyes. Cell derived fluorescence is detected for cells concentration above 25x104. Signal to background ratio is higher for DID stained cells. Analysis allowed for semi-quantitative assessment of the donor cells amount in urethras in comparison to the PBS injected control. Using the IVIS to scan thick cross sections of urethras resulted in precise localization of DID positive spots both in longitudinal and cross axes of urethra, what was not the case in regard to PKH26 dye. Microscopic analysis of histological specimens confirmed the specificity of IVIS assessment relative to DID dye. Conclusions. The IVIS system under appropriate conditions of visualization and analysis can be used as a screening method for ex vivo evaluation of transplantation procedure effects. DID can serve as fluorophore to cell labeling for this application. FATTY ACID AMIDE HYDROLASE INHIBITORS INDUCE MESENCHYMAL STEM CELL MIGRATION AND DIFFERENTIATION INTO THE OSTEOBLASTIC LINEAGE Y. Wollank1,2, R. Ramer1, A. Salamon2, K. Peters2, B. Hinz1 1 Institute of Toxicology and Pharmacology, University of Rostock, Schillingallee 70, 18057 Rostock, Germany 2 Department of Cell Biology, University of Rostock, Schillingallee 69, 18057 Rostock, Germany Migration and differentiation of mesenchymal stem cells (MSCs) are known to be involved in various regenerative processes such as bone healing. However, little is known about the pharmacotherapeutical options aiming at the mobilization and differentiation of MSCs. In view of several reports demonstrating tissue healing properties of cannabinoids, the present study focussed on inhibitors of the enzyme fatty acid amide hydrolase (FAAH) which catalyzes the degradation of endocannabinoids (anandamide [AEA]; 2-arachidonylglycerol [2-AG]) and endocannabinoid-like substances (N-oleoylethanolamine [OEA]; N-palmitoylethanolamine [PEA]). Using Boyden chamber assays, the FAAH inhibitors arachidonoyl serotonin

(AA-5HT) and URB597 were found to increase the migration of adipose-derived MSCs in a time- and concentration-dependent manner. The increase of migration by both compounds was inhibited by AM-630 (CB2 receptor antagonist) and mimicked by AEA, 2-AG, OEA and PEA. Moreover, the promigratory effect of AA-5HT and URB597 was antagonized by inhibition of the p42/44 mitogen-activated protein kinase (MAPK) pathway which became activated upon treatment with both substances. A p42/44 MAPK-dependent promigratory effect was likewise demonstrated for the selective CB2 receptor agonist JWH-133. Additional evidence for a functional effect of FAAH inhibitors on MSCs was provided by experiments demonstrating long-term stimulation with AA-5HT and URB597 to induce differentiation of MSCs into the osteoblastic lineage as evidenced by increased mineralization assessed by cresolphthalein complexone assay. Collectively, this study demonstrates FAAH inhibitors to promote the migration of MSCs via a CB2 receptor-dependent pathway involving activation of p42/44 MAPK and to induce osteoblastic differentiation. FAAH inhibitors may therefore recruit MSCs to sites of calcifying tissue regeneration and subsequently support bone regeneration via an osteoanabolic action on MSCs.

Day 3:

Invited Speakers Abstracts Identifïcation of novel factor enhacing homing of mesenchyma (stromal) stem cells to the injured tissues Adiba Isa, Postdoc, Dr.Med.Sci., Laboratory of Molecular Endocrinology (KMEB), Institute of Clinical Research, University of Southern Denmark, Odense C, Denmark MSC’s hold a great promise for tissue regeneration. Howeve, in order to exert their function, they must reach the inflamed tissues. The homing ability of MSC’s is supported by a number of observations where injected cells home to damaged sites of tissue injury. In addition, the existence of circulating MSC-like cells in the blood suggest their capacity for homing to inflammatory sites. Unfortunately, only a small number of the transplanted cells can be found in the target tissue, which is not enough to produce a therapeutic effect. This observed inefficient homing of injected MSC remains one of the major obstacles facing stromal cell therapy. In the past decade, a plethora of factors have been identified to play a key role in migration of stem cells. However, there is an urgent need to identify novel factors that could augment homing of transplanted MSC to sites of repair. Innovating Technologies to Enable Regenerative Medicine Manufacture Dr Robert J Thomas, MPharm PhD, Senior lecturer (EPSRC Early Career Fellow & Deputy Director EPSRC Centre for Innovative Manufacturing for Regenerative Medicine), Loughborough University, UK Cell based therapies present new challenges in manufacturing. Cell product quality is highly sensitive to control of process parameters, and is complicated by issues such as cell population heterogeneity, cell-cell signaling and demanding yield requirements. This increases the importance of a systematic quality by design type approach to process development and operation. The presentation will describe our approach to develop processes and technology to overcome these issues, using a series of exemplars, and focused on hematopoietic lineage commitment and erythroid progenitor proliferation. Molecular toxicity of nanoparticles using mouse bone marrow mesenchymal stem cells Dr. PV. Mohanan, PhD, FST, FASc(Aw), FSAB, Scientist & Head, Toxicology Division, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology (Govt. of India), Poojapura, Thiruvananthapuram, Kerala, India Mesenchymal stem cells are multipotent progenitor cells found in bone marrow and various tissues. Morphologically these cells have a long thin cell body and a large nucleus. These cells have the potential to differentiate as adipocytes, chondrocytes, osteoblasts and neurons under stimulated condition. The objective of this study is to exploit the mesenchymal stem cells for the molecular toxicity evaluation of nanoparticles, such as hydroxyapatite nanoparticles (HANPs) and Zinc Oxide nanoparticles (ZnONPs). Bone marrow mesenchymal stem cells (BMSC) isolated from Swiss albino mice were used for the present study. The cells were cultured and maintained in DMEM-HG medium. The isolated cells were characterized for the expression of MSCs surface markers CD44, CD90 and negative marker CD45. Confluent cells obtained after third passage were used for the evaluation of molecular toxicity. The cells were exposed to different concentration of HANPs (<50nm) and ZnONPs (~10nm). Various toxic parameters such as cytotoxicity (MTT assay), production of reactive oxygen species (ROS), apoptosis (Annexin/PI assay using FACS and caspase 3/7 activation was evaluated. The results of the study indicated that HANPs does not induce cytotoxicity up to 800µg/mL. It was also observed that oxidative stress related apoptosis and ROS production following HANPs treatment was similar to that of control. However, ZnONPs significantly affects the cellular viability in a dose dependent manner. The increased ROS production, damage of lysosomal membrane and the activation of executioner caspase-3 and caspase-7 were observed which

eventually ends in apoptosis. Further, this study also suggests that the BMSC can be used as an alternative test system for the preliminary screening of nanomaterials toxicity. Driving Regenerative Medicine Biomanufacturing Scalability through Technology Repurposing and Academic:Industry Partnerships Dr Maya Fuerstenau-Sharp, Manager R&D, Cell Culture Technologies, Sartorius Stedim Biotech, Goettingen Germany This talk highlights Sartorius Stedim's collaborative efforts to advance regenerative medicine bioprocess development. Using pluripotent stem cells to treat age-related macular degeneration Dr Amanda-Jayne Carr, Research Fellow, Institute of Ophtalmology, University College London, UK Age-related macular degeneration (AMD) is the leading cause of blindness in the western world, resulting in loss of high acuity central vision required for fine details tasks. Defects within a layer of cells in the macular region of the eye, known as the retinal pigment epithelium (RPE), result in the loss of overlying photoreceptor cells and vision loss. As a single layer of cells, the RPE is an ideal target for stem cell therapy. This talk will provide an overview of the current status and the future perspectives for AMD therapies using pluripotent stem cells.

Oral Presentation Abstracts Oral presentations will be added after the submission deadline ANALYSIS OF RELEVANT OUTCOME PARAMETRES IN PATIENTS WITH ISCHEMIC CARDIOMYOPATHY THAT PREDICT RESPONSE TO CD 133+ REGENERATIVE THERAPY Nesteruk J., Kundt G., Bubritzki S., Donndorf P., Klopsch C., Kaminski A., Steinhoff G. Reference and Translation Centre for Cardiac Stem Cell Therapy (RTC), Department of Cardiac Surgery, Medical Faculty, University of Rostock, Rostock, Germany. Address: Schilingallee 68, 18057, Rostock, Germanyn Telephone number: + 49 3814943901 Fax number: + 49 3814943909 E-mail address: iuliia.nesteruk@med.uni-rostock.de Objectives: Definition of effectiveness of intramyocardial bone marrow stem cells (BMSC) therapy is controversial, as there is no unity of results in different clinical trials. The object of the presentation is to identify relevant outcome parameters and predictors of good response to CD133+ BMSC treatment. Methods: We analyzed 150 patients with ischemic cardiomyopathy, who received intramyocardial CD133+ hematopoietic BMSC treatment combined with coronary artery bypass grafting (CABG) or CABG alone. We have stratified the patient population into responders and non-responders for 96 patients at one year follow-up after treatment. Mortality rate and MACCE up to 13 years follow-up were used to evaluate reliable study outcomes. Echocardiographic analysis, angina pectoris class (CCS), heart failure class (NYHA), injected CD133+ stem cells number, pro procedural NT-proBNP level, baseline medication and concomitant disease analysis were performed to identify surrogate end point markers and confounding factors that predict response to CD133+ BMSC treatment in congestive heart failure patients. Results: Patients with an overt heart failure (LVEF under 30% and LVEDD above 60 mm) benefit more to CD133+ cell therapy. Patients in the stem cell group improve twice as often in LV-parameters compared to control (OR, 2.72, 95% CI – 0.6-12.9, P=0.346). These functional responders who had single-step left ventricular systolic improvement (>5% LVEF) and decrease in left ventricle end diastolic diameter (-5mm LVEDD) after stem cell treatment show absence of long term mortality up to 13 years follow-up with reduced MACCE frequency. Conclusions: Combination of global ejection fraction and left ventricle end diastolic dimensions is the most reliable outcome parameter in patient responsiveness to CD133+ cell therapy and long term prognosis. Reduced ejection fraction and increased diastolic diameter are independent predictors of functional response from intramyocardial CD133+ BMSC combined with CABG in patients with ischemic cardiomyopathy.

Poster Presentation Abstracts Poster abstracts will be finalised weeks before the event CANCER STEM CELL MARKER IN CIRCULATING TUMOR CELLS OF HUMAN COLORECTAL CANCER: EXPRESSION OF CD44VARIANT EXON9 IS STRONGLY CORRELATED TO TREATMENT REFRACTORINESS, RECURRENCE, AND PROGNOSIS First Department of Surgery, University of Fukui, Japan 9101193. Takanori Goi M.D.Ph.D., Hidetaka Kurebayashi M.D., Toshiyuki Nakazawa M.D., Youhei Kimura M.D., Shigeru Katoh M.D.Ph.D., Daisuke Fujimoto M.D.,Ph.D, Mitsuhiro Morikawa M.D.,Ph.D, Kenji Koneri Yasuo M.D., Makoto Murakami M.D.Ph.D, Hirono M.D.,Ph.D., Kanji Katayama M.D.Ph.D, Akio Yamaguchi M.D.Ph.D. Background/Aim: Colorectal cancer is highly prevalent compared with other malignant tumors, and its recurrence/metastasis frequently occurs in the form of hematogenous metastasis. While the existence of circulating tumor cells (CTCs) has recently been confirmed in malignant tumors, its significance is largely unknown in patients with colorectal cancer. Recently the CD44variant exon9(CD44v9) is an important factor for cancer stem cells in colorectal cancer In the present study, we investigated the relationship between the expression of the CD44v9 in the blood and recurrence/survival rate of colorectal cancer. Materials and Methods: After peripheral blood was drawn from colorectal cancer patients, CTCs were collected. Using the Reverse Transcription-Polymerase Chain Reaction method, we examined the relationship between expression of CD44v9 mRNA and prognosis. Results: Of 150 colorectal cancer cases, 60 (40%) were high-level CD44v9 mRNA expression cases. Fifteen healthy volunteers were not detected high-level CD44v9 mRNA expression. The recurrence rate in Stage III colorectal cancer was 5 out of 35 cases (14.3%) when expression of CD44v9 was negative, and a significantly higher 8 out of 20 cases (40%) when expression was positive. Five-year survival rate was 89.5% in CD44v9 negative expression cases, and 52.4 % in CD44v9 positive expression cases, showing that survival rate was significantly poorer in the cases of positive CD44v9 expression. In Stage IV unresectable cancer patients, the 2-year survival rate was 70.1 % in cases with CD44v9 negative expression and 33.3 % in cases of CD44v9 positive expression, which was significantly worse. In the Cox proportional hazard model, CD44v9 mRNA was an independent prognostic factor. The hazard ratio for PROK1 expression was 4.445. Conclusion: Colorectal cancer with expression of CD44v9 mRNA in CTCs may have acquired drug resistance and immune evasion capability; CD44v9 mRNA is considered to be useful as a predicting factor for recurrence, prognosis, and treatment effectiveness. AN IN VITRO SYSTEM AS A TOOL ALLOWING TO EVALUATE HEMATOPOIETIC STEM CELLS LYMPHOPOIETIC CAPABILITY IN HIV INFECTED INDIVIDUALS. V. Bordoni1, D. Viola 1, M.Bibas2, A. Sacchi1, C. Agrati1,3, E. Cimini1, R. Casetti1, N. Tumino1, A. Ammassari2, A. Amendola3, F. Martini1. 1Cellular Immunology Laboratory, 2Clinical Department, 3 Virology Laboratory, National Institute for Infectious Diseases “L. Spallanzani”, Via Portuense 292, Rome Italy During HIV disease, immunological failure despite virologic success after antiretroviral treatment is associated with persistent damage to the lymphopoietic system and/or exhaustion of lymphopoiesis. These findings highlight the importance of primary hematopoietic resources in HIV pathogenesis. We analyzed the capability to differentiate toward T cells by CD34+ Hematopoietic Progenitor Cells isolated from bone marrow (BM-HPC) of 7 HIV patients, (3 naïve and 4 virological successfully ART treated). BM-HPC were cocultured until 2-3 weeks on OP9-DL1 feeder cells allowing to analyze T cells differentiation in the presence of IL-7, SCF, and FLT3ligand. The frequency of circulating HPC and the level of proinflammatory cytokines were also investigated. Our results indicate that the capability of BM-HPC to differentiate toward CD4 and CD8 T cells was impaired in patients naïve to ART in respect to those under therapy. Moreover, the level of proinflammatory cytokines released by HPC isolated from HIV naïve patients was higher in respect to those from patients under treatment. It is interesting to note that no active replication was detected in bone marrow CD34+ HPC cultures. These results suggest that an altered bone marrow environment in HIV infected patients impairs Hematopoietic Progenitor Cells differentiation toward T cells. This damage can be only partially reversed with antiretroviral therapy, contributing to the defective immune reconstitution.

TARGETED NANOPARTICULATE DELIVERY OF BIOLOGIC PAYLOADS TO NEURAL (STEM) CELLS N. Strogulski1, P. McCarron1, D. Lowry2 and S. Hawthorne1 1School of Pharmacy & Pharmaceutical Sciences, Ulster University, Cromore Road, Coleraine, United Kingdom, BT52 1SA; 2Aston Pharmacy School, Aston University, Aston Triangle, Birmingham, B4 7ET s.hawthorne@ulster.ac.uk The use of targeting nanoparticles (NPs) as delivery vehicles for specific diseases is a relatively new therapeutic area. Using specific peptides covalently attached to the periphery of the nanoparticle as targeting moieties can be an effective method for locating and gaining entry into defined cell types. Ren et al. (2014) recently formulated unlabelled PLGA nanoparticles, which were capable of delivering therapeutic payloads to astrocytes in vitro for growth inhibition demonstrating that the use of NPs for delivery of payloads to neural (stem) cells is practicable, however, the use of targeted NPs in neural (stem) cells has not been studied to any extent. Targeting of nanoparticles has been primarily confined to delivery into cancerous cell types and the lack of much published work on targeted delivery methods for NPs into neural (stem) cell types emphasises the need for the synthesis and formulation of NPS, which can be specifically targeted to a range of neural (stem) cell types. These nanoparticles could then be used for the delivery of therapeutic agents, to endogenous neural stem cells, which would stimulate the growth of new neural cells and aid in the treatment of such conditions as spinal cord injury and multiple sclerosis. In this study we generated poly(lactic-co-glycolic acid) (PLGA) nanoparticles with a payload of doxorubicin (as a model drug) and peripherally labelled with RDP peptide (the peptide on the surface of rabies virus responsible for neural cell uptake). Nanoparticulate characteristics such as size, zeta potential, entrapment efficiency, in vitro drug release profile and peptide conjugation efficiency were determined. The NPs were subsequently tested in vitro on a range on neural and non-neural (stem) cell lines and the effectiveness of their uptake and release of drug payload determined by MTT assay (doxorubicin is cytotoxic). Peptide-labelled drug loaded NPs were compared with unlabelled and empty NPs as control. Our results show there is a significant difference in the uptake of labelled NPs by neural (stem) cell types SHSY-5Y and human neural stem cells compared to non-neural HeLa cell line. Confocal microscopy images of SHSY-5Y cells confirms internalisation of the RDP-labelled nanoparticulate vehicles with doxorubicin as payload (doxorubicin fluoresces at 570 nm). These results suggest that peripheral attachment of the RDP peptide is an effective way of specifically targeting NPs to neural (stem) cell types, allowing internalisation of NPs and intracellular release of payload. THE COMPARATIVE ANALYSIS OF ACUTE MACROPHAGES INFILTRATION AFTER TRANSURETHRAL TRANSPLANTATION OF DIFFERENT CELL TYPES M. Butrym1, A. Kulesza1, W. Zarychta1, B. Dybowski2, M. Dabrowski3, L. Paczek1, A. Burdzinska1 1Department of Immunology, Transplantology and Internal Diseases, Medical University of Warsaw, Warsaw, Poland 2Department of Urology, Medical University of Warsaw, Warsaw, Poland 3Department of Large Animal Diseases with Clinic, Faculty of Veterinary Medicine, Warsaw University of Life Sciences (SGGW), Warsaw, Poland presenting author: Marta Butrym, Dept. Immunology, Transplant Medicine and Internal Diseases. Medical University of Warsaw, ul. Nowogrodzka 59, 02-006 Warsaw, Poland, corresponding author: Anna Burdzinska, Dept. Immunology, Transplant Medicine and Internal Diseases. Medical University of Warsaw, ul. Nowogrodzka 59, 02-006 Warsaw, Poland, e-mail: anna.burdzinska@wum.edu.pl Introduction. Intraurethral myogenic cell therapy is considered as a new method for treatment of urinary incontinence. Two different cell populations were proposed for such a procedure: muscle derived cells (MDCs) and mesenchymal stem cells (MSCs). The previous results in this field are promising, however poor cell survival after intramuscular transfer is a serious problem associated with this procedure. Previously it was shown that myoblasts autotransplantated into the skeletal muscle induce classical early immune reaction in the site of injection which may contribute to limited graft survival. The aim of the present study was to assess the acute infiltration of phagocytes after cell transfer into the urethral wall in large animal model. Moreover, the effect of different cell populations transplantation was evaluated in term of inflammatory infiltration induction. Material. The caprine urethras (n=4) collected 24 h after transurethral transplantation of fluorochrome labeled cells: either MDCs, MSCs, or coinjection of both cell types (MDC-MSC) or after analogous PBS injection.

Methods. The collected urethras were divided into 5-7 tissue thick cross-sections and scanned with a system for in vivo imaging in order to identify and localize transplanted cells. The samples were further proceeded with cryotome and the presence of injected cells was confirmed with fluorescent microscopy. Finally, the cross sections from all fragments of analyzed urethras were immunostained against macrophages marker - CD68 (in duplicates). The presence of macrophages was quantified using cellSens Dimension software in 2 or 3 different locations in each cross-section: 1) in the urethral lumen vicinity (in all samples); 2) randomly in the subepithelial and muscle layers (in all samples); and 3) in the proximity of transplanted cells (only in cell treated urethras). Results. In each urethra after cell transfer at least two independent clusters of grafted cells were identified (regardless of cell type injected). In control, PBS treated urethra no specific fluorescence was detected in the microscope. The quantification of macrophages in the urethral wall in relation to 3 listed locations demonstrated that, regardless of injected population, the infiltration of CD68+ cells was significantly higher around transplanted cells then around the lumen or elsewhere in the tissue. Further analysis revealed that transplantation of MSCs induced significantly smaller infiltration of macrophages in comparison to injection of either MDCs alone or in combination in MSCs (co-transplantation). Moreover, the presence of CD68+ cells close to the urethral lumen was significantly lower in PBS treated urethra in comparison to samples after injection of either MDCs or MDC-MSC, but not after administration of MSCs alone. In conclusion, presented results demonstrate that autologous transplantation of tested populations induce marked recruitment of phagocytes in the area of injection, however MSCs seem to be less immunogenic than MDCs and MDC-MSC.

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