Chiara Gomiero

Covalently bound DNA on naked iron oxide nanoparticles: Intelligent colloidal nano-vector for cell transfection

BACKGROUND Conversely to common coated iron oxide nanoparticles, novel naked surface active maghemite nanoparticles (SAMNs) can covalently bind DNA. Plasmid (pDNA) harboring the coding gene for GFP was directly chemisorbed onto SAMNs, leading to a novel DNA nanovector (SAMN@pDNA). The spontaneous internalization of SAMN@pDNA into cells was compared with an extensively studied fluorescent SAMN derivative (SAMN@RITC). Moreover, the transfection efficiency of SAMN@pDNA was evaluated and explained by computational model. METHODS SAMN@pDNA was prepared and characterized by spectroscopic and computational methods, and molecular dynamic simulation. The size and hydrodynamic properties of SAMN@pDNA and SAMN@RITC were studied by electron transmission microscopy, light scattering and zeta-potential. The two nanomaterials were tested by confocal scanning microscopy on equine peripheral blood-derived mesenchymal stem cells (ePB-MSCs) and GFP expression by SAMN@pDNA was determined. RESULTS Nanomaterials characterized by similar hydrodynamic properties were successfully internalized and stored into mesenchymal stem cells. Transfection by SAMN@pDNA occurred and GFP expression was higher than lipofectamine procedure, even in the absence of an external magnetic field. A computational model clarified that transfection efficiency can be ascribed to DNA availability inside cells. CONCLUSIONS Direct covalent binding of DNA on naked magnetic nanoparticles led to an extremely robust gene delivery tool. Hydrodynamic and chemical-physical properties of SAMN@pDNA were responsible of the successful uptake by cells and of the efficiency of GFP gene transfection. GENERAL SIGNIFICANCE SAMNs are characterized by colloidal stability, excellent cell uptake, persistence in the host cells, low toxicity and are proposed as novel intelligent DNA nanovectors for efficient cell transfection.

Repairing folding-defective α-sarcoglycan mutants by CFTR correctors, a potential therapy for limb-girdle muscular dystrophy 2D

Abstract Limb-girdle muscular dystrophy type 2D (LGMD2D) is a rare autosomal-recessive disease, affecting striated muscle, due to mutation of SGCA, the gene coding for α-sarcoglycan. Nowadays, more than 50 different SGCA missense mutations have been reported. They are supposed to impact folding and trafficking of α-sarcoglycan because the defective polypeptide, although potentially functional, is recognized and disposed of by the quality control of the cell. The secondary reduction of α-sarcoglycan partners, β-, γ- and δ-sarcoglycan, disrupts a key membrane complex that, associated to dystrophin, contributes to assure sarcolemma stability during muscle contraction. The complex deficiency is responsible for muscle wasting and the development of a severe form of dystrophy. Here, we show that the application of small molecules developed to rescue ΔF508-CFTR trafficking, and known as CFTR correctors, also improved the maturation of several α-sarcoglycan mutants that were consequently rescued at the plasma membrane. Remarkably, in myotubes from a patient with LGMD2D, treatment with CFTR correctors induced the proper re-localization of the whole sarcoglycan complex, with a consequent reduction of sarcolemma fragility. Although the mechanism of action of CFTR correctors on defective α-sarcoglycan needs further investigation, this is the first report showing a quantitative and functional recovery of the sarcoglycan-complex in human pathologic samples, upon small molecule treatment. It represents the proof of principle of a pharmacological strategy that acts on the sarcoglycan maturation process and we believe it has a great potential to develop as a cure for most of the patients with LGMD2D.

Wound-healing markers after autologous and allogeneic epithelial-like stem cell treatment

BACKGROUND AIMS Several cytokines and growth factors play an essential role in skin regeneration and epithelial-like stem cells (EpSCs) have beneficial effects on wound healing in horses. However, there are no reports available on the expression of these growth factors and cytokines after EpSC therapy. METHODS Wounds of 6 cm(2) were induced in the gluteus region of 6 horses and treated with (i) autologous EpSCs, (ii) allogeneic EpSCs, (iii) vehicle treatment or (iv) untreated control. Real time polymerase chain reaction was performed on tissue biopsies taken 1 and 5 weeks after these treatments to evaluate mRNA expression of interferon (IFN)-γ, interleukin (IL)-6, vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor (IGF)-1 and epidermal keratin (eKER). RESULTS One week after treatments, mRNA levels of IL-6 (P = 0.012) and VEGF (P = 0.008) were higher in allogeneic EpSC-treated wounds compared with controls. Also, mRNA levels of IGF-1 were higher at 1 week in both autologous (P = 0.027) and allogeneic (P = 0.035) EpSC-treated wounds. At week 5, all EpSC- and vehicle-treated wounds demonstrated significantly higher IFN-γ, VEGF and eKER mRNA expression compared with controls and compared with their respective levels at week 1. CONCLUSIONS Equine wounds treated with allogeneic EpSCs demonstrate a significant increase in mRNA expression of IL-6, VEGF and IGF-1 in the acute phase. In the longer term, an increase in IFN-γ, VEGF and eKER mRNA was detected in the wounds treated with allogenic EpSCs, autologous EpSCs or their vehicle.

Investigations of the corneal epithelium in Veterinary Medicine: State of the art on corneal stem cells found in different mammalian species and their putative application

The existence of progenitor cells that can readily differentiate into a specific cell type is a common cellular strategy for physiological tissue growth and repair mechanisms. In the mammalian cornea, many aspects regarding the nature and location of these cells are still unclear. In the human limbus (peripheral area of the cornea) progenitor cells have been found and characterized but in non-human mammals, the picture is not so clear. In this review, we examine current knowledge about the morphology of limbus and the localization of corneal epithelial stem cells in all species studied so far, comparing data with humans. We have also explored different research directions in the veterinary field in order to discuss the: i) currently used protocols and ii) best range of treatments for ocular pathologies in which corneal stem cells are involved.

A mini-review of TAT-MyoD fused proteins: state of the art and problems to solve

The transcriptional activator TAT is a small peptide essential for viral replication and possesses the property of entering the cells from the extracellular milieu, acting as a membrane shuttle. In order to safely differentiate cells an innovative methodology, based on the fusion of transcription factors and the TAT sequence, is discussed in this short review. In several studies, it has been demonstrated that TAT protein can be observed in the cell nucleus after few hours from the inoculation although its way of action is not fully understood. However, further studies will be necessary to develop this methodology for clinical purposes.

Scleraxis and MyoD fused with the TAT polypeptide may induce equine adult stem cells towards tenogenic and myogenic fates

B design and 3-D printing of scaffold with heterogeneous internal geometry is essential for cell distribution, blood vessel in growth and biomaterial degradation in bone tissue engineering. This study was designed to demonstrate the heterogeneous pores and channels in 3-D printed scaffolds for bone tissue engineering. Scaffolds were prepared using ceramic particles through 3-D printing. Pores and connecting channels with diameters of 200mm-500mm were designed for facilitating cell seeding and cell distribution. Internal pores of 50mm-200mm were designed for bone regeneration. Nano-sized surface topography was designed for enhanced degradation of scaffold. The fabricated scaffolds were evaluated using scanning electronic microscopy. SEM of fabricated scaffolds revealed that 400mm500mm inter-connecting channels crossed over the entire scaffold, that ~200 mm internal pores were scattered over the scaffold and connected to each other and to the interconnecting channels, and that ~200 nm pores showed on the surfaces of inter-connecting channels and internal pores, which would play an important role in increasing the surface ratio of materials and facilitating material degradation. A heterogeneous profile of connecting channels and internal pores was evident in these 3-D printed biomimetic scaffolds. As a conclusion, the biomimetic design and fabrication of scaffolds for bone tissue engineering can be fulfilled by a 3-D printing process. Heterogeneous profiles of inter-connecting channels, internal pores, and nano-sized surface topography can be generated to provide a biomimetic environment suitable for bone tissue engineering.Background: Myocardial infarction (MI) was the leading cause of death in worldwide. MicroRNAs (miRNAs) regulate gene expression at the post-transcriptional level and are known to play essential roles in various aspects of biological processes, including cell viability, proliferation, development and differentiation. The purpose of this study was to investigate difference of miRNA profiles between infarct zone and border zone in post-MI remodeling using the second generation sequencing.Neia Naldaiz-Gastesi1, Patricia Garcia-Parra1, Maria Goicoechea1, Sonia Alonso-Martin2, Ana Aiastui1, Macarena Lopez-Mayorga3, Paula Garcia-Belda4, Jaione Lacalle1,5, Veronique Le Berre6, Ander Matheu1, Jose Manuel Garcia-Verdugo4, Jaime J. Carvajal3, Frederic Relaix2, Adolfo Lopez de Munain1 and Ander Izeta1 1Instituto Biodonostia, Spain 2Myology Research Center, France 3Centro Andaluz de Biologia del Desarrollo, Spain 4Instituto Cavanilles, Universidad de Valencia, Spain 5University of the Basque Country (UPV-EHU), Spain 6UMR INSA, FranceOur knowledge of the regenerative ability of the auditory system is still inadequate. Moreover, new treatment techniques for hearing impairment using cochlear implant and tissue engineering, call for further investigations. Tissue engineering and regenerative strategies have many applications ranging from studies of cell behavior to tissue replacement and recently there have been significant advances in the biotechnological tools followed by development of new interventions, including molecules, cells, and even biodegradable biomaterials. This thesis presents results of tissue engineering approaches used in vitro with the long-term aim of facilitating auditory nerve and spiral ganglion regeneration. The first part describes the use of neurotrophic factors and neurosteroids for promoting survival and growth of nerve cells and the second part describes the effective usage of a biotechnology method, microcontact imprinting technique, to control key cellular parameters modifying chemical cues on the surface. The failure of the spiral ganglion neurons to regenerate was postulated to be due to the limited capacity of neurons to re-grow axons to their target. In paper I, we focused our studies on the role of GDNF in promoting spiral ganglion neuron outgrowth. The effect of three neurotrophins, among them GDNF, on spiral ganglion neurons in vitro was evaluated. The neuronal outgrowth was characterized by light microscopy and immunohistochemistry. The results speak in favor of GDNF, which promoted neuronal growth and branching, and Schwann cell alignment along the neurons in culture. The study support the role of GDNF as a potent factor, exerted neurogenic effects on cochlear cells in a degree dependent on the concentration used, confirming the hypothesis of GDNF being an oto-protector for chemicaland noiseinduced hearing loss and potential drug candidate for the inner ear. This might be relevant for future regenerative therapies and could have implications for tissue engineering techniques. In the second study, paper II, the objective was similarly to evaluate the efficacy of dendrogenin, a neurosteroid analogue, which can be applied to the cochlea. Dendrogenin was also tested in the presence and absence of other growth factors and the effect on adult neural stem cells was investigated. The study showed that neural stem cells exhibited proliferation/differentiation responses. Based on fluorescent labeling and a sphere-formation assay, we observed that adult neural stem cells induced proliferation. We asked whether the stem cells would differentiate into the major cell types of the nervous system and mainly neurons. Thus, neurotrophic supplement was added to the culture medium and was shown to have a selective effect on outgrowth of neuronal population. β3-tubulin positive neurons with BrdU positive nuclei were found and similar to other studies, we observed that the rate of differentiation increased with declining of BrdU expression. We found that despite the ongoing neuronal differentiation, there was an apparent difference of the neuronal outgrowth among the spheres treated with dendrogenin. The newly formed neurons were not found to send long projections into the local circuitry and the total cell number and length remained limited. Taken together, the protocols described inhere provide a robust tool to expand the biological role of dendrogenin that was in favor of differentiation when added to neuronal cell lines. The results of this study add new knowledge and better understanding of the possible action of dendrogenin in regenerative therapy. In paper III a strategy to guide spiral ganglion neurons was developed using a microcontact technique. The surface for neuronal guidance was designed with favorable extracellular proteins to promote the neurite outgrowth. Micro-contact imprinting provided a versatile and useful technique for patterning the guidance surface. Imprinting generated a patterned surface in a controllable, predictable, and quantifiable manner. A range of events followed the patterning including alignment, polarity and directionality was reported and observed by microscopic description. The dynamic microenvironment that resulted from the synergistic combination of extracellular guidance cues and Schwann cells selectively instructed and directed the terminal extension of neurons into unior bi-polar fate. In summary, applying new factors such as molecules, cells and surfaces provides unique possibilities to recruit spiral ganglion neurons into their regenerative ability. Additionally, creating an environment that incorporates multiple molecular and cellular cues will offer exciting opportunities for elucidating the mechanisms behind nerve regeneration and highlight specific considerations for the future tissue engineering. LIST OF PUBLICATIONS This thesis is based on the following original papers, which will be referred to in the text by their Roman numerals. I. Marja Bostrom, Shaden Khalifa, Henrik Bostrom, Wei Liu, Ulla Friberg, Helge Rask-Andersen. Effects of neurotrophic factors on growth and glial cell alignment of cultured adult spiral ganglion cells. Audiology Neurootology 2010; 15(3): 175-186. II. Shaden AM Khalifa, de Medina Philippe, Sandrine Silvente-Poirot, Anna Erlandsson, Hesham ElSeedi and Marc Poirot. The novel steroidal alkaloids dendrogenin A and B promote proliferation of adult neural stem cells. Under revision in Biochemical and Biophysical Research Communications. III. Shaden AM Khalifa, Per Bjork, Christian Vieider, Mats Ulfendahl, and Eric Scarfone. Neuronal Polarity Mediated by Micro-scale Protein Patterns and Schwann Cells in vitro. Tissue Engineering and Regenerative Medicine 2013; 10(5): 266-272. CONTENTS Abstract List of publications Abbreviations Chapter 1: Introduction 1 1.1 Ear anatomy 1 1.2 Hearing physiology 6 1.3 Hearing Loss 8 1.4 Cochlear implant 9 1.5 Tissue engineering strategies 11 Aims 18 Chapter 2: Materials and methods 19 2.1 Ethical permission and animal care 19 2.2 Tissue dissection 19 2.3 Micro-pattern fabrication 20 2.4 Culture procedure 22 2.5 Immunohistochemistry 24 2.6 Imaging 25 2.7 Time Lapse Video 26 2.8 Imaging analysis 26 2.9 Axon analyzer software 27 2.10 Statistical Analysis 27 Chapter 3: Results 28 3.1 Neurotrophins stimulate neuronal regeneration in vitro 28 3.2 Dendrogenin activity on adult neural stem cells 29 3.3 Protein patterning 30 3.4 Neuronal polarity 30 3.5 Cells in culture 31 Chapter 4: Discussion 33 4.1 GDNF effects on spiral ganglion cells in vitro 33 4.2 Dendrogenin effects on adult neural stem cells in vitro 34 4.3 Patterning proteins 35 4.4 Neuronal guidance and polarity 35 Chapter 5: Conclusions and future prospective 37 5.1 Conclusions 37 5.2 Prospective 38 Acknowledgments 40 References 43 LIST OF ABBREVIATIONSS cells have been recognized as a potential tool to restore cells damaged by cerebral ischemic injury. Key functions such as the replacement of neural cells have been recently challenged by intrinsic bystander capacities of undifferentiated donor cells. One of opportunity for neurological disorder treatment is the transplantation of mesenchymal stem cells (MSCs) which have neuroprotective, neuroregenerative and anti-inflamatory properties. However, a comprehensive knowledge how transplanted MSCs exert their therapeutic achievements is still lacking. The aim of the project was to analyze the presence, distribution and quantity of human bone marrow mesenchymal stem cells (hBM-MSCs) transplanted into focal brain ischemic rats. The experiments were performed in adult male Wistar rats withbrain focal ischemiainduced with 1μl/50nmol ouabain(sodium-potassium pump inhibitor) injection into right stratium. Then 5x105 hBM-MSC (Lonza) stained with iron nanoparticles and rhodamine (Molday, BioPAL) were transplanted into internal carotid artery, 48 hours after brain insult. At 1, 3, 7 and 14 days rat brains were removed. Immunocytochemical analysis of human markers using different antibodies anti: CD44, STEM121and Ku80 were performed. The preliminary results showed that after intra-arterially injection of hBM-MSC, the donor cells were present in the ipsilateral rat hemisphere between cortical cortex and stratium near the ischemic lesion. The positive staining for Molday particles and human antigens were observed at 1, 2, 3 and 7 days after hBM-MSC transplantation. The further studies relating to the function of transplanted cells are in progress.M stem cells (MSCs) represent a population of multipotent stem cells with immunomodulatory, antiapoptotic and cytoprotective capabilities and thus hold a great promise for treatment of many inflammatory diseases and for use in a regenerative medicine. Numerous studies have shown that the administration of MSCs in combination with an immunosuppressive drug prolongs allograft survival in comparison with use of MSCs or the drug alone. However, the exact mechanism of such synergism has not yet been described.

Biomimetic innovative application for tendon and skin regenerative medicine.

B design and 3-D printing of scaffold with heterogeneous internal geometry is essential for cell distribution, blood vessel in growth and biomaterial degradation in bone tissue engineering. This study was designed to demonstrate the heterogeneous pores and channels in 3-D printed scaffolds for bone tissue engineering. Scaffolds were prepared using ceramic particles through 3-D printing. Pores and connecting channels with diameters of 200mm-500mm were designed for facilitating cell seeding and cell distribution. Internal pores of 50mm-200mm were designed for bone regeneration. Nano-sized surface topography was designed for enhanced degradation of scaffold. The fabricated scaffolds were evaluated using scanning electronic microscopy. SEM of fabricated scaffolds revealed that 400mm500mm inter-connecting channels crossed over the entire scaffold, that ~200 mm internal pores were scattered over the scaffold and connected to each other and to the interconnecting channels, and that ~200 nm pores showed on the surfaces of inter-connecting channels and internal pores, which would play an important role in increasing the surface ratio of materials and facilitating material degradation. A heterogeneous profile of connecting channels and internal pores was evident in these 3-D printed biomimetic scaffolds. As a conclusion, the biomimetic design and fabrication of scaffolds for bone tissue engineering can be fulfilled by a 3-D printing process. Heterogeneous profiles of inter-connecting channels, internal pores, and nano-sized surface topography can be generated to provide a biomimetic environment suitable for bone tissue engineering.Background: Myocardial infarction (MI) was the leading cause of death in worldwide. MicroRNAs (miRNAs) regulate gene expression at the post-transcriptional level and are known to play essential roles in various aspects of biological processes, including cell viability, proliferation, development and differentiation. The purpose of this study was to investigate difference of miRNA profiles between infarct zone and border zone in post-MI remodeling using the second generation sequencing.Neia Naldaiz-Gastesi1, Patricia Garcia-Parra1, Maria Goicoechea1, Sonia Alonso-Martin2, Ana Aiastui1, Macarena Lopez-Mayorga3, Paula Garcia-Belda4, Jaione Lacalle1,5, Veronique Le Berre6, Ander Matheu1, Jose Manuel Garcia-Verdugo4, Jaime J. Carvajal3, Frederic Relaix2, Adolfo Lopez de Munain1 and Ander Izeta1 1Instituto Biodonostia, Spain 2Myology Research Center, France 3Centro Andaluz de Biologia del Desarrollo, Spain 4Instituto Cavanilles, Universidad de Valencia, Spain 5University of the Basque Country (UPV-EHU), Spain 6UMR INSA, FranceOur knowledge of the regenerative ability of the auditory system is still inadequate. Moreover, new treatment techniques for hearing impairment using cochlear implant and tissue engineering, call for further investigations. Tissue engineering and regenerative strategies have many applications ranging from studies of cell behavior to tissue replacement and recently there have been significant advances in the biotechnological tools followed by development of new interventions, including molecules, cells, and even biodegradable biomaterials. This thesis presents results of tissue engineering approaches used in vitro with the long-term aim of facilitating auditory nerve and spiral ganglion regeneration. The first part describes the use of neurotrophic factors and neurosteroids for promoting survival and growth of nerve cells and the second part describes the effective usage of a biotechnology method, microcontact imprinting technique, to control key cellular parameters modifying chemical cues on the surface. The failure of the spiral ganglion neurons to regenerate was postulated to be due to the limited capacity of neurons to re-grow axons to their target. In paper I, we focused our studies on the role of GDNF in promoting spiral ganglion neuron outgrowth. The effect of three neurotrophins, among them GDNF, on spiral ganglion neurons in vitro was evaluated. The neuronal outgrowth was characterized by light microscopy and immunohistochemistry. The results speak in favor of GDNF, which promoted neuronal growth and branching, and Schwann cell alignment along the neurons in culture. The study support the role of GDNF as a potent factor, exerted neurogenic effects on cochlear cells in a degree dependent on the concentration used, confirming the hypothesis of GDNF being an oto-protector for chemicaland noiseinduced hearing loss and potential drug candidate for the inner ear. This might be relevant for future regenerative therapies and could have implications for tissue engineering techniques. In the second study, paper II, the objective was similarly to evaluate the efficacy of dendrogenin, a neurosteroid analogue, which can be applied to the cochlea. Dendrogenin was also tested in the presence and absence of other growth factors and the effect on adult neural stem cells was investigated. The study showed that neural stem cells exhibited proliferation/differentiation responses. Based on fluorescent labeling and a sphere-formation assay, we observed that adult neural stem cells induced proliferation. We asked whether the stem cells would differentiate into the major cell types of the nervous system and mainly neurons. Thus, neurotrophic supplement was added to the culture medium and was shown to have a selective effect on outgrowth of neuronal population. β3-tubulin positive neurons with BrdU positive nuclei were found and similar to other studies, we observed that the rate of differentiation increased with declining of BrdU expression. We found that despite the ongoing neuronal differentiation, there was an apparent difference of the neuronal outgrowth among the spheres treated with dendrogenin. The newly formed neurons were not found to send long projections into the local circuitry and the total cell number and length remained limited. Taken together, the protocols described inhere provide a robust tool to expand the biological role of dendrogenin that was in favor of differentiation when added to neuronal cell lines. The results of this study add new knowledge and better understanding of the possible action of dendrogenin in regenerative therapy. In paper III a strategy to guide spiral ganglion neurons was developed using a microcontact technique. The surface for neuronal guidance was designed with favorable extracellular proteins to promote the neurite outgrowth. Micro-contact imprinting provided a versatile and useful technique for patterning the guidance surface. Imprinting generated a patterned surface in a controllable, predictable, and quantifiable manner. A range of events followed the patterning including alignment, polarity and directionality was reported and observed by microscopic description. The dynamic microenvironment that resulted from the synergistic combination of extracellular guidance cues and Schwann cells selectively instructed and directed the terminal extension of neurons into unior bi-polar fate. In summary, applying new factors such as molecules, cells and surfaces provides unique possibilities to recruit spiral ganglion neurons into their regenerative ability. Additionally, creating an environment that incorporates multiple molecular and cellular cues will offer exciting opportunities for elucidating the mechanisms behind nerve regeneration and highlight specific considerations for the future tissue engineering. LIST OF PUBLICATIONS This thesis is based on the following original papers, which will be referred to in the text by their Roman numerals. I. Marja Bostrom, Shaden Khalifa, Henrik Bostrom, Wei Liu, Ulla Friberg, Helge Rask-Andersen. Effects of neurotrophic factors on growth and glial cell alignment of cultured adult spiral ganglion cells. Audiology Neurootology 2010; 15(3): 175-186. II. Shaden AM Khalifa, de Medina Philippe, Sandrine Silvente-Poirot, Anna Erlandsson, Hesham ElSeedi and Marc Poirot. The novel steroidal alkaloids dendrogenin A and B promote proliferation of adult neural stem cells. Under revision in Biochemical and Biophysical Research Communications. III. Shaden AM Khalifa, Per Bjork, Christian Vieider, Mats Ulfendahl, and Eric Scarfone. Neuronal Polarity Mediated by Micro-scale Protein Patterns and Schwann Cells in vitro. Tissue Engineering and Regenerative Medicine 2013; 10(5): 266-272. CONTENTS Abstract List of publications Abbreviations Chapter 1: Introduction 1 1.1 Ear anatomy 1 1.2 Hearing physiology 6 1.3 Hearing Loss 8 1.4 Cochlear implant 9 1.5 Tissue engineering strategies 11 Aims 18 Chapter 2: Materials and methods 19 2.1 Ethical permission and animal care 19 2.2 Tissue dissection 19 2.3 Micro-pattern fabrication 20 2.4 Culture procedure 22 2.5 Immunohistochemistry 24 2.6 Imaging 25 2.7 Time Lapse Video 26 2.8 Imaging analysis 26 2.9 Axon analyzer software 27 2.10 Statistical Analysis 27 Chapter 3: Results 28 3.1 Neurotrophins stimulate neuronal regeneration in vitro 28 3.2 Dendrogenin activity on adult neural stem cells 29 3.3 Protein patterning 30 3.4 Neuronal polarity 30 3.5 Cells in culture 31 Chapter 4: Discussion 33 4.1 GDNF effects on spiral ganglion cells in vitro 33 4.2 Dendrogenin effects on adult neural stem cells in vitro 34 4.3 Patterning proteins 35 4.4 Neuronal guidance and polarity 35 Chapter 5: Conclusions and future prospective 37 5.1 Conclusions 37 5.2 Prospective 38 Acknowledgments 40 References 43 LIST OF ABBREVIATIONSS cells have been recognized as a potential tool to restore cells damaged by cerebral ischemic injury. Key functions such as the replacement of neural cells have been recently challenged by intrinsic bystander capacities of undifferentiated donor cells. One of opportunity for neurological disorder treatment is the transplantation of mesenchymal stem cells (MSCs) which have neuroprotective, neuroregenerative and anti-inflamatory properties. However, a comprehensive knowledge how transplanted MSCs exert their therapeutic achievements is still lacking. The aim of the project was to analyze the presence, distribution and quantity of human bone marrow mesenchymal stem cells (hBM-MSCs) transplanted into focal brain ischemic rats. The experiments were performed in adult male Wistar rats withbrain focal ischemiainduced with 1μl/50nmol ouabain(sodium-potassium pump inhibitor) injection into right stratium. Then 5x105 hBM-MSC (Lonza) stained with iron nanoparticles and rhodamine (Molday, BioPAL) were transplanted into internal carotid artery, 48 hours after brain insult. At 1, 3, 7 and 14 days rat brains were removed. Immunocytochemical analysis of human markers using different antibodies anti: CD44, STEM121and Ku80 were performed. The preliminary results showed that after intra-arterially injection of hBM-MSC, the donor cells were present in the ipsilateral rat hemisphere between cortical cortex and stratium near the ischemic lesion. The positive staining for Molday particles and human antigens were observed at 1, 2, 3 and 7 days after hBM-MSC transplantation. The further studies relating to the function of transplanted cells are in progress.M stem cells (MSCs) represent a population of multipotent stem cells with immunomodulatory, antiapoptotic and cytoprotective capabilities and thus hold a great promise for treatment of many inflammatory diseases and for use in a regenerative medicine. Numerous studies have shown that the administration of MSCs in combination with an immunosuppressive drug prolongs allograft survival in comparison with use of MSCs or the drug alone. However, the exact mechanism of such synergism has not yet been described.