Die Inzidenz von neurodegenerativen Erkrankungen und Schlaganfällen steigt in Folge der Überalterung der westlichen Gesellschaft immer weiter an. Die Behand-lung von Schlaganfall-, Alzheimer und Parkinsonpatienten ist bisher aber meist unbefriedigend bzw. weitgehend erfolglos. Ein neues Modell in der Schlaganfallforschung wurde daher am Schaf entwickelt. In diesem wird auch der in den letzten zwei Jahrzehnten verstärkt verfolgte zelltherapeutische Ansatz untersucht (BOLTZE et al. 2011, DREYER et al. 2012). Neurale Vorläuferzellen gelten dabei, auf Grund ihrer wichtigen Rolle bei den endogenen Reparaturmechanismen nach einem Schlaganfall, als besonders vielversprechend. Die Gewinnung dieser Zellen für eine autologe Transplantation ist jedoch aufwendig und nur eingeschränkt möglich. Im Vergleich zu Nervengewebe stellt die Haut eine sowohl beim Tier als auch beim Menschen leicht zugängliche und in ausreichendem Maß verfügbare Quelle verschiedener Stamm- und Vorläuferzellen dar. Bei verschiedenen Spezies wurde die Isolation spezieller, dermaler Vorläuferzellen beschrieben, die als skin-derived precursor cells (SKPs) bezeichnet werden. SKPs wiesen dabei ein ähnliches Differenzierungspotential auf wie neurale Vorläuferzellen (TOMA et al. 2001, FERNANDES et al. 2006). Ein Einsatz der SKPs in der Schlaganfalltherapie wäre somit denkbar, muss aber zunächst im Schafmodell erforscht werden. SKPs wurden jedoch noch nicht bei der Spezies Schaf isoliert. Ziel der vorliegenden Arbeit war es daher, ein Isolationsprotokoll für SKPs aus der ovinen Dermis zu etablieren und diese morphologisch und immunzytologisch zu charakterisieren. Im Rahmen dieser Arbeit wurden verschiedene in der Literatur beschriebene Isolati-onsverfahren an ovinen Hautproben getestet und modifiziert. Es wurden verschiedene Körperregionen auf ihre Eignung zur Probenentnahme und zur anschließenden Isolierung untersucht. Des Weiteren wurde der Effekt einer Rasur eine Woche vor Exzision des Hautareals auf die Sphäroidbildung überprüft. Der Einsatz von Enzymen in Kombinationslösungen oder singulär wurde variiert und eine unterschiedlich intensive mechanische Aufbereitung der Proben durchgeführt. Der Erfolg der zwei vielversprechendsten Isolationsprotokolle wurde statistisch validiert. Außerdem wurde der Effekt einer initialen Fibronektinbeschichtung analysiert. Die von den isolierten Zellen gebildeten sphärenartigen Zellaggregate wurden unter morphologischen Gesichtspunkten sechs und neun Wochen nach Isolation ausgewertet. Dabei wurden die Anzahl der Sphäroide/cm², die Größe und die Form berücksichtigt. Des Weiteren erfolgte eine immunzytologische Analyse der Sphäroide mit Fokus auf das in der Literatur beschriebene Expressionsmuster von SKPs und neuralen Vorläuferzellen. Für die Isolation von ovinen SKPs erwies sich die Regio nasofrontalis als das geeignetste Hautareal. Dabei war die Isolation eine Woche nach Rasur des beprobten Areals zuverlässiger als ohne diese. Bei vergleichender Betrachtung der Methoden erwies sich ein enzymatisch orientiertes Isolationsverfahren modifiziert nach FERNANDES und MILLER (2009) als zielführend. Neben einer hohen Anzahl an isolierten Zellen erfolgte in jedem Versuchsdurchgang eine Zusammenlagerung der Zellen in frei flotierenden Aggregaten. Diese waren im Median 70,97 µm groß. Auf Grund ihrer Geometrie ist es korrekter sie als Sphäroide und nicht, wie bei anderen Spezies üblich, als Sphären zu bezeichnen. Eine anfängliche Beschichtung der Zellkulturplatten mit Fibronektin hatte keinen fördernden Effekt auf die Bildung und die Größe der Sphäroide. Lediglich eine anfänglich höhere Proliferationsrate war bemerkbar. Immunzytologisch konnte gezeigt werden, dass in den Sphäroiden eine heterogene Zellpopulation vorlag. Die Sphäroide wurden überwiegend von Zellen gebildet, in denen neben mesenchymalen Markern auch klassische Vorläuferantigene wie Nestin und Sox2 nachgewiesen wurden. Das immunzytologische Expressionsmuster ist damit vergleichbar mit dem von SKPs anderer Spezies. Außerdem wurden in unterschiedlicher Ausprägung Antigene detektiert, die typischerweise in neuralen Vorläuferzellen der ventrikulären und subventrikulären Zone vorkommen. Dies konnte auch in den Positivkontrollen für das ovine Gehirn bestätigt werden. Die Anzahl proliferierender Zellen in den Sphäroiden war relativ gering und die Anzahl an kokultivierter Keratinozyten minimal. Die Zusammenfassung der heterogenen Vorläuferzellpopulation unter dem Begriff skin-derived precursor cells ist auf Grund ihres dermalen Ursprungs und ihrer morphologischen und immunzytologischen Eigenschaften gerechtfertigt. Somit ist es in dieser Arbeit gelungen, zum ersten Mal SKPs aus der ovinen Dermis zu isolieren und über neun Wochen zu kultivieren. Es wurde ein Isolationsprotokoll entwickelt, das eine Sphäroidbildung reproduzierbar ermöglicht und an die Gegebenheiten beim Schaf angepasst ist. Bevor eine autologe Transplantation von diesen SKPs etwa im Schlaganfallmodell am Schaf vorgenommen werden kann, ist eine intensivere Untersuchung der isolierten Zellen etwa mittels PCR durchzuführen und eine fluoreszenzbasierte Zellsortierung der heterogenen Vorläuferzellen zu entwickeln. / In consequence of the demographic changes in modern western society, the inci-dence of neurodegenerative diseases and stroke is increasing. Unfortunately, there is still no successful or at least satisfactory treatment available for patients who suffer from stroke Alzheimer’s or Parkinson’s disease. Therefore, a new animal model in stroke research has been established in sheep (BOLTZE et al. 2011, DREYER et al. 2012). First cell therapy studies have already been performed in this model. Especially neural precursor cells seem to be promising as they play an important role in endogenous repair processes in the brain after stroke. However, the extraction of these cells prior to an autologous transplantation is elaborate and of limited success. Compared to neural tissue, skin is an easily accessible and sufficiently available source of a variety of stem and precursor cells in animals as well as in humans. Thus, the isolation of a specific type of dermal precursor cells, called skin-derived precursor cells (SKPs), seems to be easier compared to neural precursor cells and in vitro SKPs are capable of neural differentiation as well (TOMA et al. 2001, FERNANDES et al. 2006). According to these findings, a therapeutic application of SKPs after stroke seems to be promising. Prior to that, however, intensive studies in the ovine stroke model are necessary. Thus, SKPs have to be isolated from the dermis of sheep for an autologous transplantation. Therefore, the aim of this dissertation has been the establishment of an optimal isolation protocol for SKPs from the ovine dermis as well as the morphological and by immunocytochemical characterisation of those cells. Within this study, several previously described isolation protocols were modified for ovine skin. Skin samples were taken from several body regions to assess the local suitability for excision and isolation. Additionally, the effect of shaving the areas one week before sampling on spheroid forming was tested. A variety of enzymes was used alone and in combination. Furthermore, the effectiveness of an isolation protocol using enhanced mechanical treatment was analysed. The two most promising protocols were evaluated statistically and compared to each other. In these experiments, the influence of an initial fibronectin coating was determined as well. The isolated cells formed spheroids, which were assessed after six and nine weeks of cultivation considering the amount of spheroids per cm², their size and form. Moreover, immunocytochemical tests were conducted, focusing on expression patterns described for SKPs and neural precursor cells. According to these experiments, it is advisable to take skin samples from the naso-frontal region one week after shaving. Comparing all tested protocols, a predominantly enzymatic isolation protocol modified according to FERNANDES and MILLER (2009) was most successful. A high cell yield was achieved and free-floating spheroids formed spontaneously in all test runs. The median diameter of these spheroids was 70.97 µm. Due to their three-dimensional shape, it is more correct to use the term “spheroid” instead of the commonly used term “sphere”. Growing the isolated cells initially on fibronectin coated culture plates does not support both formation and size of the spheroids. Only a higher cell proliferation at the beginning of cultivation can be noticed. Immunocytochemical assays demonstrated that the formed spheroids consisted of a heterologous cell population. Besides mesenchymal antigens the cells in the spheroids expressed characteristic antigens of precursor cells, like Nestin and Sox2. Thus, the immunocytochemical expression pattern is comparable to SKPs isolated from other species. Furthermore, common markers of neural precursor cells of the ventricular and subventricular zone, whose existence in the ovine brain was also proven in this study, were detected in the spheroid forming cells. There were only a few proliferating cells and a minimal amount of keratinocytes in the spheroids. Due to the dermal origin and the given morphological and immunocytochemical characteristics, the heterogeneous cell population can be addressed by the term “skin-derived precursor cells”. In conclusion, in this study ovine SKPs were isolated for the first time and cultured successfully over nine weeks. An isolation protocol was established, which guarantees reproducible formation of spheroids in cell isolates from ovine dermis. Further intensive examinations of the isolated cells, for example using PCR, have to be conducted before SKPs can be applied in autologous transplantation in the ovine stroke model. Additionally, the usage of fluorescence-activated cell sorting of the heterogeneous precursor cells should be considered.

Our lab has recently found that E2F3, an essential cell cycle regulator, regulates the self-renewal capacity of neural precursor cells (NPCs) in the developing mouse brain. Chromatin immunoprecipitation (ChIP) and immunoblotting techniques revealed several E2F3 target genes, including the polycomb group (PcG) protein, EZH2. Further ChIP and immunoblotting techniques identified the neural stem cell self-renewal regulators p16INK4a and Sox2 as shared gene targets of E2F3 and PcG proteins, indicating that E2F3 and PcG proteins may co-regulate these target genes. E2f3-/- NPCs demonstrated dysregulated expression of EZH2, p16INK4a, and SOX2 and decreased enrichment of PcG proteins at target genes. Restoring EZH2 expression to E2f3+/+ levels restores p16INK4a and SOX2 expression levels to near E2f3+/+ levels, and also partially rescues NPC self-renewal capacity toward E2f3+/+ levels. Taken together, these results suggest that E2F3 controls NPC self-renewal by modulating expression of p16INK4a and SOX2 via regulation of PcG expression, and potentially PcG recruitment.

Made available in DSpace on 2016-06-02T20:21:22Z (GMT). No. of bitstreams: 1 2665.pdf: 1669132 bytes, checksum: dfe7f18cc9241784c82420b0409bd47e (MD5) Previous issue date: 2009-09-21 / Universidade Federal de Sao Carlos / Disintegrins are toxins commonly found in snake venoms whose biological effects occour upon interaction with surface receptors known as integrins. DisBa-01, an RGD disintegrin isolated from a cDNA library made with mRNAs from the venom gland of Bothrops alternatus, bears anti-metastatic, anti-angiogenic and anti-thrombotic activity in nude mice, partially mediated by interaction with integrin _v_3. Studies with Wistar rats also show that protein has angiogenic activity during regeneration after surgery. The objectives of this study were to evaluate the effects of DisBa-01 on adhesion, deadhesion and proliferation of C2C12 myoblasts and evaluate the electrophoretic profile of secreted proteins of DisBa-01-treated cells. Myoblasts (103 cells/well) were incubated with five concentrations of DisBa-01 under different incubation times and adhesion substracts (plastic, collagen type I or fibronectin). The supernatant was collected for 2DE analysis and the cells were quantified. Also, flow cytometry was used to evaluate the expression of integrins (_2, _4, _5, _v, _v_3, _1 and _4). DisBa-01 inhibited adhesion to fibronectin in high concentration (1000nM) and to collagen type I in all tested concentrations, but did not detach cells from collagen or fibronectina matrix nor affected myoblast proliferation. Flow cytometry showed that integrins _v and _1 were present. 2DE analysis points to an increase of secreted proteins, mainly in higher DisBa- 01 concentration (1000nM). In conclusion, C2C12 myoblasts are sensitive to DisBa-01, suggesting this protein initiates a sinalization cascade mediated by integrins, which modifies protein expression and secretion. / Desintegrinas são toxinas frequentemente encontradas em venenos de serpentes e cujos efeitos biológicos ocorrem devido à interação com receptores de superfície conhecidos como integrinas. A DisBa-01, uma desintegrina RGD isolada de mRNAs da glândula venenífera de Bothrops alternatus, possui atividade anti-metastática, anti-angiogênica e anti-trombótica em camundongos atímicos, parcialmente mediada pela interação com a integrina _v_3. Estudos in vivo com ratos Wistar também indicam que esta proteína pode ter atividade pró-angiogênica durante regeneração pós-cirúrgica. O objetivo deste estudo foi avaliar os efeitos da DisBa-01 na adesão, desadesão e proliferação celular de mioblastos C2C12, além de avaliar o perfil eletroforético das proteínas secretadas por estas células sob condições normais e tratadas. As células (103 células/poço) foram incubadas com diferentes concentrações de DisBa-01 em diferentes tempos e substratos (plástico, colágeno tipo I e fibronectina). O meio de cultura foi recolhido para análise por eletroforese bidimensional (2DE) e as células foram quantificadas. Além disso, foi realizada citometria de fluxo para avaliar a expressão de integrinas (_2, _4, _5, _v, _v_3, _1 e _4). A DisBa-01 inibiu a adesão das células à fibronectina em alta concentração (1000nM), além de inibir a adesão das células ao colágeno em todas as concentrações testadas, mas não promoveu a desadesão de células aderidas a estes dois substratos tampouco afetou significativamente a proliferação de mioblastos. A análise por citometria de fluxo identificou as integrinas _v e _1, corroborando em parte os resultados obtidos. A análise por 2DE do sobrenadante indicou que a DisBa-01 induz a um aumento no número de proteínas secretadas, principalmente na maior concentração testada (1000nM). Em conclusão, os mioblastos C2C12 são sensíveis à DisBa-01, sugerindo que esta proteína inicia uma cascata de sinalização celular mediada por integrinas que modifica a secreção protéica.

Our lab has recently found that E2F3, an essential cell cycle regulator, regulates the self-renewal capacity of neural precursor cells (NPCs) in the developing mouse brain. Chromatin immunoprecipitation (ChIP) and immunoblotting techniques revealed several E2F3 target genes, including the polycomb group (PcG) protein, EZH2. Further ChIP and immunoblotting techniques identified the neural stem cell self-renewal regulators p16INK4a and Sox2 as shared gene targets of E2F3 and PcG proteins, indicating that E2F3 and PcG proteins may co-regulate these target genes. E2f3-/- NPCs demonstrated dysregulated expression of EZH2, p16INK4a, and SOX2 and decreased enrichment of PcG proteins at target genes. Restoring EZH2 expression to E2f3+/+ levels restores p16INK4a and SOX2 expression levels to near E2f3+/+ levels, and also partially rescues NPC self-renewal capacity toward E2f3+/+ levels. Taken together, these results suggest that E2F3 controls NPC self-renewal by modulating expression of p16INK4a and SOX2 via regulation of PcG expression, and potentially PcG recruitment.

Gene mit gerigem Einfluss auf einen untersuchten Phänotyp können durch den Ein- schluss einer genetischen Variation im Tierversuch untersucht werden. Adulte Neuro- genese, der Prozess der Neubildung und Integration von funktionellen Neuronen in das existierende neurale Netzwerk, wird von vielen solchen Genen mit geringem Effekt beeinflusst. All diese Gene im lebenden Tier zu untersuchen wäre mit einem hohen Arbeitsaufwand verbunden, und würde hohe Tierzahlen erfordern. Bereits publizierte Ergebnisse zeigen, dass diese Gene auch in der Zellkultur unter Verwendung von Zelllinien genetisch rekombinanter Tiere untersucht werden können (Kannan et al., 2016). Die hier verwendeten, ingezüchteten Mausstämme des so genannten BXD Panels stellen die Nachkommen der Kreuzung der beiden Mausstämme C57BL/6J und DBA/2J dar (Peirce et al., 2004), die sich in der Ausprägung von unterschiedlichen Neurogenese bezogenen Phänotypen bereits deutlich unterscheiden (Kempermann et al., 2006). Durch die Verwendung der BXD Tiere wird hierbei die Aussagekraft der genetischen Variation mit dem Zellkultursystem verbunden. Die Aussagekraft dieser Studie ist jedoch darin limitiert, dass aufgrund des verwendeten Protokolls nur eine Zelllinie pro Mausstamm generiert werden konnte. Daher präsentiere ich hier ein neues Protokoll welches es erlaubt eine Zelllinie aus nur einem einzelnen Tier zu generieren. Diese Methode kombiniert zwei bestehende Zellkultursysteme, die Neurosphärenkultur und die Monolayerkultur. Es stellte sich heraus, dass die Überlebensrate der einzelnen Zelllinien vom biologischen Hintergrund der Zellen beeinflusst wird. So ist die Überlebensrate von Zellen der DBA/2J Mäuse deutlich schlechter als die der C57BL/6J oder die der F1 Generation aus der Verpaarung der beiden Stämme. Es zeigte sich allerdings, dass diese Überlebensrate nicht ausschließlich von der vorhandenen Anzahl proliferierender Zellen abhängt, da B6D2F1 (F1 Generation mit einem C57BL/6J Muttertier) signifikant weniger proliferierende (Ki67 positive) Zellen in vivo aufweisen, jedoch keine geringere Überlebensrate der Zelllinien haben. Eine hoch standardisierte, umfangreiche Analyse der Zelllinien aller vier Mausstämme (C57BL/6J, DBA/2J, und die zwei reziproken F1 Nachkommen BDF1 und DBF1) zeigte eine hohe Varianz innerhalb genetisch identischer Linien, was die Be- stimmung eines Effektes, der durch den genetischen Hintergrund der Linien verursacht wird, beeinträchtigte. Die Zelllinien werden signifikant von äußeren Faktoren beeinflusst, wie z.B. durch das Einfrieren der Zellen. Dies gibt Hinweise darauf, dass Untersuchungen in der Zellkultur genau geplant, kritisch hinterfragt, sowie möglichst alle potentiellen Einflussfaktoren gleich gehalten werden müssen. Nur so können valide, aussagekräftige Ergebnisse mit der Zellkultur gewonnen werden. Automatische Zellkultursysteme, neue Mikroskopieverfahren, sowie besser definierte Langzeitstudien werden unser Verständnis von Zellen in der Zellkultur deutlich verbessern und dabei ihren Wert, sowie bestehende Limitationen, endgültig klären.:List of Figures I List of Tables II List of Abbreviations III List of Publications V 1. Introduction 1 1.1 Genetic variation in animal research 2 Recombinant inbred strains 3 The BXD panel 4 The Gene Network 5 Genetic modifications 5 
 1.2 Adult hippocampal neurogenesis 6 History 7 Clinical relevance 8 The BXD panel and adult hippocampal neurogenesis 9 
 1.3 Developmental stages of neural precursor cells 9 
 1.4 Studying adult neurogenesis in vitro 11 Culturing hippocampal precursor cells 11 A mouse cell culture genetic reference panel 13 
 1.5 Tracking 13 
 1.6 Objectives 15 
 2. Materials and Methods 16 2.1 Components and equipment 16 
 2.2 Antibodies 20 
 2.3 Recipes 21 
 General buffers and solutions 21 Cell culture solutions 21 Immunocytochemistry solutions 23 Immunohistochemistry solutions 24 
 2.4 Experimental animals 25 
 2.5 Cell culture 25 Coating of cell culture vessels 25 Fire-polished pipettes 25 Dentate gyrus isolation 26 Neurosphere assay 26 Monolayer culture 27 
 2.6 Immunocytochemistry 29 BrdU staining preparations 29 Staining protocol 30 Imaging and counting 30 
 2.7 Immunohistochemistry 30 Sample preparation 30 Staining protocol 31 Cell counting 31 
 2.8 Tracking 32 Cell preparation and imaging setup 34 Image processing 35 Data analysis 35 
 2.9 Generation of CRISPR/Cas mediated knock-out lines 36 Construct design and cloning 36 E. coli Top10 transformation and plasmid isolation 37 Transfection of neural precursor cells and expansion of knock-out lines 38 Genotyping of the generated cell lines 39 Agarose gel electrophoresis 40 
 2.10 Statistical analysis 40 2.11 Data visualization 40 3. Results 41 3.1 Single animal monolayer cultures41 The three phenotypes of the neurosphere assay 44 Neurosphere assay phenotypes could not predict the survival of a cell line 45 The genetic background had an influence on all three phenotypes of the neurosphere assay 46 Significantly less proliferating cells in vivo but no difference in the neurosphere assay of BDF1 compared to BL6 animals 48 BDF1 cells could not be activated to form more spheres but sphere size could be increased using KCl 49 
 3.2 A new cell line phenotyping standard operation procedure and its application 50 Line generation data 52 Marker staining 54 Cell tracking 55 
 3.3 Cell culture – a system with limitations 59 Freezing effect 60 Cell culture data - technical variance hinders the analysis of small effects 62 3.4 Migration speed and GFAP 63 The strength of the BXD panel – cumulative data 65 
 3.5 Other applications of the tracking procedure 68 
 Tracking labeled cells in an embryonic zebrafish xenograft model 68 Cell tracking in mouse retina explants 68 4. Discussion 70 4.1 Single animal monolayer cultures – a new protocol 70 
 4.2 A new phenotyping pipeline 74 
 4.3 Semi-automated (user-supervised) cell tracking 77 
 4.4 A possible correlation between migration speed and differentiation 79 4.5 CRISPR/Cas knock-out lines - an ill-conceived system with high potential 82 4.6 The problem of the validity of cell culture experiments - a comment 83 4.7 Conclusion 84 
 Bibliography 88 A Single animal cell line generation protocol 106 
 B Cell line characterization SOP 112 
 C R Scripts 117 / Uncovering gene loci that assert only small effects onto a phenotype of interest, can be achieved by including genetic variation in animal research. Adult hippocampal neurogenesis, the process of the formation of new neurons and their functional integration into existing circuitry, is influenced by a broad range of such small effect genes. Analyzing all of these genes in vivo would be laborious and require a high number of animals. Previously published data merged the power of genetic variation with a cell culture system by using cell lines generated from the BXD recombinant inbred mouse strains (Kannan et al., 2016). These strains are inbred progeny of F2 crosses originating from the two mouse strains C57BL/6J and DBA/2J (Peirce et al., 2004), which already differ quite extensively in neurogenesis related phenotypes (Kempermann et al., 2006). As previous studies were limited by the number of strains that could be generated due to the demand for high numbers of animals, I developed a new method that allows the generation of a cell line from one single animal. For this new method, I combined the neurosphere culture with a subsequent monolayer culture. The survival of the resulting cell lines, is thereby greatly influenced by the genetic background. The survival rate of cell lines derived from DBA/2J animals is much lower as compared to C57BL/6J-derived lines or lines from the F1 generation of crossing the two strains. Whether or not a cell line survived did not seem to be solely influenced by the number of proliferating cells in vivo, as B6D2F1 (F1 progeny with a C57BL/6J mother) showed significantly less proliferative (Ki67 positive) cells in vivo while exhibiting a survival rate that exceeded both parental strains. An extensive study of the cell lines gained from all four mouse strains (C57BL/6J, DBA/2J, and the two reciprocal F1 progeny B6D2F1 and D2B6F1) in a highly standardized manner showed that the individual difference between single cell lines was rather high, hampering the successful detection of in-between strain differences. The standardized characterization of the generated cell lines, further allowed the identification of external factors, influencing the cells, as for example the freezing of the cells. This indicates that cell culture experiments need to be thoroughly planned and critically scrutinized, while all external factors should be kept as constant as possible to ensure the validity of the resulting data. Automated cell handling, new imaging technologies, as well as more defined long-term studies will greatly improve the understanding of cells in culture and thereby show their true values and limitations.:List of Figures I List of Tables II List of Abbreviations III List of Publications V 1. Introduction 1 1.1 Genetic variation in animal research 2 Recombinant inbred strains 3 The BXD panel 4 The Gene Network 5 Genetic modifications 5 
 1.2 Adult hippocampal neurogenesis 6 History 7 Clinical relevance 8 The BXD panel and adult hippocampal neurogenesis 9 
 1.3 Developmental stages of neural precursor cells 9 
 1.4 Studying adult neurogenesis in vitro 11 Culturing hippocampal precursor cells 11 A mouse cell culture genetic reference panel 13 
 1.5 Tracking 13 
 1.6 Objectives 15 
 2. Materials and Methods 16 2.1 Components and equipment 16 
 2.2 Antibodies 20 
 2.3 Recipes 21 
 General buffers and solutions 21 Cell culture solutions 21 Immunocytochemistry solutions 23 Immunohistochemistry solutions 24 
 2.4 Experimental animals 25 
 2.5 Cell culture 25 Coating of cell culture vessels 25 Fire-polished pipettes 25 Dentate gyrus isolation 26 Neurosphere assay 26 Monolayer culture 27 
 2.6 Immunocytochemistry 29 BrdU staining preparations 29 Staining protocol 30 Imaging and counting 30 
 2.7 Immunohistochemistry 30 Sample preparation 30 Staining protocol 31 Cell counting 31 
 2.8 Tracking 32 Cell preparation and imaging setup 34 Image processing 35 Data analysis 35 
 2.9 Generation of CRISPR/Cas mediated knock-out lines 36 Construct design and cloning 36 E. coli Top10 transformation and plasmid isolation 37 Transfection of neural precursor cells and expansion of knock-out lines 38 Genotyping of the generated cell lines 39 Agarose gel electrophoresis 40 
 2.10 Statistical analysis 40 2.11 Data visualization 40 3. Results 41 3.1 Single animal monolayer cultures41 The three phenotypes of the neurosphere assay 44 Neurosphere assay phenotypes could not predict the survival of a cell line 45 The genetic background had an influence on all three phenotypes of the neurosphere assay 46 Significantly less proliferating cells in vivo but no difference in the neurosphere assay of BDF1 compared to BL6 animals 48 BDF1 cells could not be activated to form more spheres but sphere size could be increased using KCl 49 
 3.2 A new cell line phenotyping standard operation procedure and its application 50 Line generation data 52 Marker staining 54 Cell tracking 55 
 3.3 Cell culture – a system with limitations 59 Freezing effect 60 Cell culture data - technical variance hinders the analysis of small effects 62 3.4 Migration speed and GFAP 63 The strength of the BXD panel – cumulative data 65 
 3.5 Other applications of the tracking procedure 68 
 Tracking labeled cells in an embryonic zebrafish xenograft model 68 Cell tracking in mouse retina explants 68 4. Discussion 70 4.1 Single animal monolayer cultures – a new protocol 70 
 4.2 A new phenotyping pipeline 74 
 4.3 Semi-automated (user-supervised) cell tracking 77 
 4.4 A possible correlation between migration speed and differentiation 79 4.5 CRISPR/Cas knock-out lines - an ill-conceived system with high potential 82 4.6 The problem of the validity of cell culture experiments - a comment 83 4.7 Conclusion 84 
 Bibliography 88 A Single animal cell line generation protocol 106 
 B Cell line characterization SOP 112 
 C R Scripts 117

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24839号 / 医博第5007号 / 新制||医||1068(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 井上, 治久, 教授 寺田, 智祐, 教授 林, 康紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Yes / Expression of the transmembrane NG2 chondroitin sulphate proteoglycan (CSPG) defines a distinct population of NG2-glia. NG2-glia serve as a regenerative pool of oligodendrocyte progenitor cells in the adult central nervous system (CNS), which is important for demyelinating diseases such as multiple sclerosis, and are a major component of the glial scar that inhibits axon regeneration after CNS injury. In addition, NG2-glia form unique neuron–glial synapses with unresolved functions. However, to date it has proven difficult to study the importance of NG2-glia in any of these functions using conventional transgenic NG2 ‘knockout’ mice. To overcome this, we aimed to determine whether NG2-glia can be targeted using an immunotoxin approach. We demonstrate that incubation in primary anti-NG2 antibody in combination with secondary saporin-conjugated antibody selectively kills NG2-expressing cells in vitro. In addition, we provide evidence that the same protocol induces the loss of NG2-glia without affecting astrocyte or neuronal numbers in cerebellar brain slices from postnatal mice. This study shows that targeting the NG2 CSPG with immunotoxins is an effective and selective means for killing NG2-glia, which has important implications for studying the functions of these enigmatic cells both in the normal CNS, and in demyelination and degeneration.

Hyperthermia damages both developing and adult brains, especially when it occurs after ischemia or stroke. Work presented in this dissertation used in vitro models of these stresses to investigate mechanisms underlying damage to immature neurons and neural precursors cultured from embryonic rat brain. Studies described in Chapter 2 investigated the effects of a brief, intense hyperthermic stress (30-45 min at 43ºC). This stress produced a selective depletion of nestin-immunoreactive neural precursor cells, and reduced proliferation, as evidenced by reduced BrdU incorporation into young Tuj1-immunoreactive neurons. The stress activated caspase 3, and produced multiple signs of nuclear damage as well as early and persisting mitochondrial depolarization. Cycloheximide, an inhibitor of protein synthesis, reduced cell death. All these findings suggest an apoptotic death process. Studies described in Chapter 3 used a combination of oxygen-glucose deprivation (OGD, 2 h) followed by mild 41ºC hyperthermia for 90 min (T). The combined OGDT stress reduced both survival in monolayer cultures and colony-forming ability in neurospheres. Cell death occurred gradually over 2 days, and was accompanied by caspase activation that began within 6 h post-stress. Post-stress application of cycloheximide or a general caspase inhibitor (especially qVD-OPH) reduced cell death, but specific inhibitors of caspases 2, 3, 8 or 9 were ineffective. OGDT led to upregulation of the pro-apoptotic protein Bim as well as redistribution of Bax from cytoplasm to mitochondria within 6 h. Persisting mitochondrial depolarization began within 3 h following the combined OGDT stress, but not following individual OGD or T stresses alone. These findings suggest that OGD sensitizes neural precursor cells to hyperthermia-induced damage, and that the combined OGDT stress kills neural precursors via apoptotic mechanisms that include activation of mitochondrial death pathways. Results of these studies suggest that immature neurons and neural precursors are especially vulnerable to hyperthermia-induced damage via apoptotic mechanisms. Pan-caspase inhibitors may be a promising therapeutic strategy to preserve viability of these cells following stroke with hyperthermia.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on July 1, 2010). Includes bibliographical references.

Submitted by Nadir Basilio (nadirsb@uninove.br) on 2018-06-18T22:38:09Z No. of bitstreams: 1 Erika Cassia Barroso Batista.pdf: 1153764 bytes, checksum: d2f3d36a962ecb178963820d00e754fa (MD5) / Made available in DSpace on 2018-06-18T22:38:09Z (GMT). No. of bitstreams: 1 Erika Cassia Barroso Batista.pdf: 1153764 bytes, checksum: d2f3d36a962ecb178963820d00e754fa (MD5) Previous issue date: 2015-12-16 / The activation, proliferation and migration of myogenic precursor cells are essential for muscle regeneration after injury, orchestrated by cells and local components, mainly inflammatory cells, especially macrophages. These are identified as a target for the treatment of muscle injuries. Laser therapy has shown good results in treatment of injuries and the ability to accelerate the migration of various cell types, but there are no reports on their effect on macrophage products influencing the migration of myogenic precursor cells. The aim of the study was to evaluate the effect of low level laser (LLL) on migration of myoblasts cultured with macrophage culture supernatants of different phenotypes. Therefore, C2C12 cells were cultured with supernatants from cultures of macrophages (J774) treated with LPS and IFN-γ (for the activation phenotype M1), IL-4 (M2a phenotype) and IL-10 and dexamethasone (M2c phenotype) and LLL irradiated at wavelengths of 660nm and 780nm (70mW; 17,5J / cm2; 14.3 s; 0,8J). Supernatants from macrophage cultures were harvested 24h after irradiation and transferred to myoblast cultures. Migration was assessed using the scratch assay and the results statistically analyzed. Myoblasts cultured with phenotype macrophage supernatants M2c and irradiated with LBP (660nm) showed higher migration capability that cultured with supernatants of M2C phenotype of macrophages after 12 hours of cultivation. There was no difference between the other groups. The LLL was able to modulate the migration of myoblasts C2C12 when M2c supernatants of macrophage phenotype / A ativação, proliferação e migração das células precursoras miogênicas são essenciais na regeneração muscular após lesões, orquestrados pelas células e componentes locais, principalmente pelas células inflamatórias, em especial os macrófagos. Estes são apontadas como alvo para o tratamento das lesões musculares. A laserterapia tem demonstrado bons resultados no tratamento destas lesões e na capacidade de acelerar a migração de vários tipos celulares, mas não existem relatos sobre seu efeito nos produtos de macrófagos que influenciam a migração de células precursoras miogênicas. O objetivo do estudo foi avaliar o efeito do laser de baixa potência (LBP) sobre a migração de mioblastos cultivadas com sobrenadantes de culturas de macrófagos de diferentes fenótipos. Para tanto, as células C2C12 foram cultivadas com sobrenadantes de culturas de macrófagos (J774) tratadas com LPS e IFN- γ (ativação para o fenótipo M1), IL-4 (fenótipo M2a) e IL-10 e dexametasona (fenótipo M2c) e irradiadas com LBP nos comprimentos de onda de 660nm e 780nm (70mW; 17,5J/cm2; 14,3 s; 0,8J). Os sobrenadantes das culturas de macrófagos foram colhidos 24h após as irradiações e transferidos para culturas de mioblastos. A migração foi avaliada por meio do ensaio de ferida e os resultados submetidos à análise estatística. Após 12h de cultivo, os mioblastos cultivados com sobrenadantes de macrófagos de fenótipo M2c e irradiados com LBP (660nm) mostraram maior capacidade de migração que os cultivados com sobrenadantes de macrófagos de fenótipo M2c não irradiados. Não houve diferença entre os demais grupos. O LBP foi capaz de modular a migração de mioblastos C2C12 quando cultivados com sobrenadantes de macrófagos de fenótipo M2c.