11^th International Conference on Tissue Engineering & Regenerative Medicine October 18-20, 2018 Hotel: Holiday Inn Rome Aurelia, Rome, Italy

Stepanova O V works in the field of the Regenerative Medicine. She studies the resident stem/progenitor cardiac and neural cells. She demonstrated that the heart of the patients with heart failure due to dilated cardiomyopathy contains the progenitor resident cardiomyocytes and dedifferentiated cardiomyocytes. These cardiac cells possibly can proliferate and differentiate to mature cardiomyocytes and recover heart function and structure after injury. She found that myosin activating protein kinases may contribute in myofibril formation during the cardiomyocyte differentiation. Now she is actively working in the field of investigation of olfactory mucosa neural stem/progenitor and ensheathing cells. Development of methods for obtaining the cell cultures from the olfactory mucosa and the study of the therapeutic efficacy of these cells in experimental posttraumatic spinal cord cysts will create the preconditions for their successful application in the treatment of patients with posttraumatic cysts of spinal cord

Statement of the Problem: The pathological processes developing after spinal cord injuries often result in the formation of cysts. Treatment of posttraumatic spinal cord cysts is a difficult task, and the existing surgical and medical methods are ineffective. One of the most promising and emerging trends is cell therapy. The most perspective type of cells may be the ensheathing cells of the olfactory mucosa. However, the therapeutic effect of these cells on posttraumatic cysts of the spinal cord remains unexplored. The purpose of this research is to study the therapeutic effect of rat olfactory ensheathing cell transplantation on posttraumatic cysts of the spinal cord. Methodology & Theoretical Orientation: The ensheathing cells were obtained from rat olfactory mucosa by our modified procedure and were characterized by the expression of markers p75NTR and GFAP detected by immunofluorescence method. The study of the therapeutic effect of the transplantation on posttraumatic cysts was conducted on a model developed in our laboratory. The formation of cysts was confirmed by MRI. The cells were transplanted in different amounts (750000, 1500000). The dynamic of the recovery of motor activity of the hind limbs of rats was assessed using the BBB test. Findings: The improvement of the motor activity after transplantation of the olfactory ensheathing cells into posttraumatic cysts was demonstrated. The necessary number of cells (1.5 million) for the transplantation was also established, and a neuroprotective effect of a given number of cells was revealed. Conclusion & Significance: For the first time the therapeutic effect of rat olfactory ensheathing cell transplantation on posttraumatic cysts was demonstrated. Further research in this area will help to develop a combined use of the cell therapy by olfactory ensheathing cells with surgical and drug therapy for the treatment of patients with posttraumatic cysts of the spinal cord.

Hyo Jin Kang has his professional experiences in the Department of Neurosurgery, Seoul National University Hospital Clinical Research Institute (~2007), Department of Urology, Yonsei University Medical Center Research Center (~2015), Department of Biological Sciences, Yonsei University College of Medicine (~2017) and Department of Plastic and Reconstructive Surgery, Seoul National University Bundang Hospital (~2018). He has his teaching experiences in the Department of Life Science, Gachon University (2012 ~ 2014).

Chronic inflammation and impaired neovascularization are a critical factor contributing to delayed wound healing in diabetic patients. To overcome the therapeutic limitations of diabetic wounds (DBW), we investigated the effects of catechol-functionalized hyaluronic acid (HA-CA) patch combined with adipose-derived mesenchymal stem cells (ADSCs) in diabetic wound mouse models. The diabetic mice (DB, C57BL/6) were induced with streptozotocin (50 mg/kg, < 250 mg/dL) and divided into 4 groups; control (DBW), DBW+ADSCs, DBW+HA-CA, DBW+HA-CA+ADSCs (n=10 each). The ADSCs were labeled with fluorescent cell tracer PKH26 to detect the mesenchymal stem cells (MSCs) migration. The wound area was evaluated by ImageJ and was assessed for collagen content, thickness and vascularity of granulation tissue, apoptosis, re-epithelialization. The angiogenesis was evaluated by immunohistochemistry (von Willebrand Factor and CD31) and real-time PCR. The DBW healings were improved in all groups compared with control, especially in DBW+HA-CA+ADSCs group. In histopathologic scoring about edema, inflammation and congestion, the score significantly decreased in DBW+HA-CA+ADSCs group. PKH26-labeled ADSCs were observed in the subcutaneous tissue and muscle layer of the wounds. Apoptotic cells by TUNEL staining were decreased in all groups compared with control. In angiogenesis assay, the expressions of vWF and CD31 were significantly increased in DBW+HA-CA+ADSCs group. The HA-CA combined with ADSCs represents a promising approach to improve DBW healing by re-epithelialization and promoting angiogenesis. HA-CA combined with ADSCs will be a new strategy for clinical treatment.

Cheng-Wen Wu is an Academician of Academia Sinica, and Distinguished Chair Professor of National Yang-Ming University in Taiwan. He was a pioneer in biophysical approach to molecular biology, best known for his work on presenting biophysical evidence of the 4-step mechanism of gene transcription, and the discovery of the presence of zinc ions in eukaryotic transcription factor TFIIIA, the first example for the class of proteins known as zinc finger proteins. After returning to Taiwan, he made many important contributions to the studies of cancer metastasis in lung cancer, and of acute and chronic lung injuries such as ARDS and COPD, two of the serious health threats to the Taiwanese population, with important clinical significances. Recently, he is interested in applying stemness concept for analyzing tumor progression in lung cancer as well as gene and stem cell therapies of lung injuries. He has published over 264 scientific articles in international journals and has won numerous prestigious scientific awards

Lung diseases, such as acute respiratory distress syndrome (ARDS) or chronic obstructive pulmonary disease (COPD), are both major public health problems but currently without any effective pharmacologic approach for the treatment. Stem cell therapy based on transplantation of in vitro propagated stem/progenitor cells has been proposed as a potential solution to restore lung functions. However, due to the complexity of cell source and lung microenvironment, whether transplanted cells have differentiated for reconstitution of airway/alveolar epithelium were questioned. Furthermore, safety issues have raised concerning the use of stem cells in vivo. Although past gene therapy studies using viral vectors in human has been hampered due to adverse effects and safety issues, recent clinical trials have shown remarkable therapeutic benefits and an excellent safety record with improved vector designs. Our lab has focused on in vivo gene delivery of stemness genes in somatic lung epithelial cells using PEI nanoparticles for lung injury treatment. In a mouse model of elastase-induced emphysema, we found that the transient gene delivery of COPX-1 in alveolar epithelial cells post-injury induced efficient regeneration of alveolar epithelium and improved pulmonary function. The regenerated regions showed normal alveolar epithelial phenotype and extracellular matrix components, without the symptoms of neoplasia. Our study suggests that in vivo delivery of stemness genes in somatic cells in pathologic loci is a feasible approach for tissue regeneration. The target cells could proliferate and differentiate more efficiently due to the native identity and microenvironment. In vivo gene delivery may hold promise for the future treatment of lung diseases such as ARDS or COPD.

Marie Cuvellier started her PhD after completing her degree in Pharmaceutical Sciences at the Faculty of Pharmacy of Rennes I, and an MSc in Human Toxicology from the University of Paris Descartes. Her work at the IRSET focuses on the development of 3D multicellular liver models through 3D bioprinting, while working on the implementation of this technology at a larger scale within the institute.

The use of conventional 2D in vitro culture models is increasingly questioned and their ability to accurately mimic the complete physiological function of the organs or tissues of interest can be limited. For a few times now, 3D models have been successfully developed by the team Dymec of IRSET, with improved function and phenotypes of many cells type, including primary and transformed hepatic cells. The project aims to improve those 3D models by building a multicellular organoid-like liver model, with an organized architecture and in a reproducible and high-throughput way. We relied on extrusion based bioprinting technology, which allows the precise and spatially controlled deposition of a biological material containing cells to form definite microarchitectures. The first step of the work was to define the printing parameters: hydrogel concentrations, step of cross linking, printing temperature, pressure and speed, have been optimized to obtain a printable structure which remained stable over the long term. Secondly, the long-term biocompatibility of these structures was tested by encapsulation of transformed hepatic cells. They preserved their long-term viability (>30 days) and were able to proliferate in this biomatrix optimized for bioprinting. They organized themselves into spheroids/acini-like structures and displayed an apico-basal polarity after seven days of culture. The next step aim was to improve the liver function of the created structures, by bioprinting an architecturally organized co-cultures with parenchymal cells, by adding to the currents bioprinted structures non-parenchymal cells.

Sajan George is an enthusiastic researcher in infectious diseases, cancer and stem cell biology. He has functioned as a Veterinarian and Clinical Researcher in industry as well. He has been involved in phase II clinical trials of anti-fungal drugs in canines and pharmacokinetic testing of liposomal anti-cancer drugs in non-human primates. His research towards the comparison of Mycobacterium spp. harboring dairy cows’ and in vitro infected macrophages has unveiled the adaptations of pathogen for survival and virulence. His Master’s thesis has identified the effect of feed antibiotics on weanling piglets using functional genomics and proteomics approaches. His Doctoral research activity is differentiation of adipose stem cells (ASCs) to functional neurons using low-level laser irradiation.

Statement of the Problem: Neurological diseases and disorders pose a challenge for treatment and rehabilitation due to the limited capacity of nervous system to repair. Autologous adipose stem cells (ASCs) therapy is considerably safe and effective for clinical applications to treat nervous system disorders. Low intensity laser irradiation (LILI) acts by altering cellular signaling cascade, stimulating cellular processes and changing transcriptional modalities causing ASCs to differentiate to other cell-types. The focus of this study is the differentiation of ASCs to functional neuronal cells using LILI in the presence of cell growth factors. Methodology & Theoretical Orientation: The effect of light on living systems and cells, referred to as photobiomodulation (PBM), is attributed to the generation of reactive oxygen species (ROS). ASCs were isolated from healthy human subjects undertaking abdominoplasty in clinics, proliferated as neurospheres and induced to differentiate into neurons in the presence of near infra-red (NIR) irradiation. Findings: We were able to explore specific energy levels of PBM capable of generating ROS and inducing cellular biophysiological processes towards neuronal-lineages. Laser-induced differentiated neurons were isolated from the mixed population and subjected to functional analysis. Evidently, ASCs treated with growth factors and LILI possess better survivability and are capable of neuronal differentiation, which makes them an ideal choice for replacement therapies in clinics. Conclusion & Significance: Estimating the mechanistic changes in ASCs during its transformation to other lineages allow us finding solutions to maladies with patient’s own genetic background. This study gave us indications on how universal forces such as NIR (light) can manipulate the fate of a living cell in favor of growth, division, and/or differentiation. Further, it enables us in exploring methodologies for modulating signaling pathways and metabolic circuitry towards multipotency in mesodermal cells.

Elena Butoi graduated from Faculty of Physics, University of Bucharest and obtained her PhD in Biological Sciences in 2008 at ICBP-NS. Since 2014, she is Scientific Researcher Grade I at ICBP-NS and Head of Cell Adhesion Laboratory in the Biopathology and Therapy of Inflammation Department. Her group focuses on the effects of cross-talk between immune cells and vascular cells in progression of atherosclerosis and other cardiovascular disorders in normal or diabetic conditions. She has published more than 28 papers in ISI journals. Since 2017 she is involved in an interesting project aiming to uncover relevant diabetes-related alterations in aortic valves by developing in vitro 3D-printed model of aortic valve leaflet seeded with human valvular cells.

Statement of the Problem: Calcific aortic valve disease (CAVD) is the third leading cause of cardiovascular disease being a progressive process, with initial valvular endothelium inflammation, followed by fibrotic thickening and extensive calcification of the valve leaflets. Currently, pharmacological interventions for slowing down the progression of CAVD are unavailable. Therefore, physiologically suitable in vitro models are required to study the cellular and molecular mechanisms of CAVD. Aim of this study is to obtain a 3D construct with a structure similar with the aortic valve leaflet. Materials & Methods: Human valvular interstitial cells (VIC) were encapsulated in bioink and bioprinted. Printing was performed with the 3DDiscovery® platform (regenHU, Switzerland) and several bioink formulations. Valvular endothelial cells (VEC) were seeded on the construct surface. The constructs were maintained in culture for 7-21 days. Evaluation of viability of cells was determined using live/dead and MTT cell viability assays. Cell phenotype was analyzed by immunohistochemistry using phalloidin and specific antibodies. Results & Discussions: In the current study, we obtained a 3D bioprinted construct with human VIC inside and a VEC layer on the top. Encapsulated VIC developed a well-defined cell network within construct and express low levels of alpha-smooth muscle actin (α-SMA) compared with VIC in bi-dimensional culture. The Western blot data indicated that bioprinted VICs have a less activated phenotype than those grown in bi-dimensional culture. VEC seeded on the constructs adhere, proliferate and form a monolayer at surface of the construct exhibiting endothelial specific markers: PECAM-1 and von Willebrand factor. Conclusions: We have obtained 3D bioprinted constructs with valve leaflet structure, with viable VIC encapsulated in constructs and VEC on the surface. The obtained structure will help for further investigations of the dynamic interaction between valve interstitial and endothelial cells, in order to study the valvular disease progression and to develop potential therapeutic interventions.

The effect of the 3D calcium phosphate (CaP) matrix on the vital activity of leukemic T-lymphoblastoid Jurkat cells was studied upon their co-cultivation with multipotent mesenchymal stromal cells (MMSC) using the methods of flow cytometry and the Cell-IQ system for real-time intravital imaging. Titanium substrates with a diameter of 10-12 mm and carrying a microarc coating with a surface roughness factor Ra 2-5 μm were used as matrix-stimulus. Fourteen-day co-cultivation of Jurkat cells with MMSC (2D co-culture) resulted in expression levels increase of early and late activation molecules (CD25+ and CD95+) on leukemia cells in comparison to the control group (2D Jurkat culture). Similar changes were observed when 3D matrix was added to the Jurkat cell culture. In the case of the Jurkat + MMSC + 3D Matrix culture, the number of CD95+ Jurkat cells decreased (by 25.3%), and CD25+ increased (by 1.5 times) in comparison to the 2D culture of Jurkat + MMSC. The Cell-IQ visualization system revealed negative dynamics (in comparison to 2D co-cultivation) in Jurkat cells population during co-cultivation with MMSC in the presence of 3D matrix, as well as an increase in adhesion capability and motor activity (amoeboid-like motions) of large (apt, polyploid) Jurkat cells. Alizarin red staining (calcified intercellular matrix) significantly increased in the 21-day 2D and 3D MMSC cultures in the presence of Jurkat cells. Morphofunctional interaction of MMSC and Jurkat, modulated with a rough CaP matrix, requires detailed interpretation for further use if the fields of regenerative medicine and bioengineering of bone tissue under conditions of tumor growth

The influence of the activator (microspheres with conjugated CD3, CD2 and CD28 MKAT) on Jurkat cell’s vital activity, during co-culturing with MMSC under the effect of three dimensional (3D) calcium phosphate (CaP) matrix. The results were recorded using flow cytometry and the Cell-IQ system of intravital imaging. CaP coating with a roughness index Ra of 2-5 μm was applied to titanium substrates of 10×10*1 mm in size and used during the experiment. Expression levels of early activation CD25+ molecules and late activation CD95+ molecules on leukemia cells decreased 2-fold for CD25+ and by 25% for CD95+ compared to the control group (2D culture Jurkat) during co-cultivation of T-cells Jurkat with multipotent mesenchymal stem cells (MMSC) (3D co-culture) for 14 days with an addition of the activator. Large activated Jurkat T-cells, capable of adhering to plastic, were detected using Cell-IQ imaging system, which in presence of 3D-CP matrix; increase their motor activity in comparison to negative dynamics of cell proliferation. It should be noted that color intensity and the number of focal points of alizarin red staining significantly increase during co-culturing of activated Jurkat with MMSC after 21 days of growth, which suppose the aspiration of the MMSC osteodifferentiation processes in both 2D and 3D cultures. Obtained data can serve as a basis for deciphering interaction mechanisms of MMSC with activated immunocompetent tumor cell lines in the processes of implant osseointegration with a variety of CaP coatings.