Zugl.: Giessen, Univ., Diss., 2008 / Text engl.

Zugl.: Giessen, Univ., Diss., 2008

Stammzellen mit ihrer besonderen Fähigkeit sich selbst zu erneuern und zu differenzieren stellen einen faszinierenden Zelltyp für Grundlagenforschung und angewandte Wissenschaften dar. Pluripotente embryonale Stammzellen (ES Zellen), die aus Zellen der inneren Zellmasse von Präimplantationsembryonen etabliert werden, können ekto-, meso- und endodermale Zelltypen sowie Keimzellen hervorbringen. Im Gegensatz dazu sind multipotente adulte Stammzellen in ihrem Entwicklungspotential eingeschränkt, sie differenzieren sich zu allen Zelltypen ihres Gewebes. Zum Beispiel hämatopoetische Stammzellen (HSZs), die sich in Blut-bildenden Geweben wie dem Knochenmark befinden, vermögen sich in alle Blutzellen zu differenzieren. Während der Differenzierung von Stammzellen ändert sich nicht deren Genom, sondern ihre epigenetische Regulation. Durch epigenetische Mechanismen werden Zelltypen mit verschiedensten Phänotypen und Funktionen generiert. Für Stammzelltherapien ist ein tieferes Verständnis des Zusammenhangs von Epigenom und zellulärer Funktion wichtig. Im Rahmen dieser Dissertation war es mein Ziel, differenzierende Stammzellkulturen auf ihre Genexpression, ihre Chromatinregulation und ihr Differenzierungspotiential hin zu analysieren. Um Histonmodifikationen, die einen möglichen Mechanismus epigenetischer Regulation darstellen, global untersuchen zu können, sind zunächst, durchusszytometrische Protokolle etabliert worden, die die Analyse einzelner Zellen ermöglichen sollten. Mit dieser Methode konnten reduzierte Levels von Histonazetylierung in differenzierten ES Zellen gezeigt werden. Im Gegensatz dazu beobachtete ich vergleichbare Levels von Histonazetylierung in unreifen und reifen Knochenmarkzellen. Zusätzlich untersuchte ich die Wirkung des Histondeazetylase-Inhibitors (HDI) Trichostatin A (TSA) auf Knochenmarkzellkulturen, in denen auch HSZs enhalten sind. Nach Behandlung mit TSA erhöhte sich der Anteil von Zellen mit in vitro und in vivo hämatopoetischer Aktivität, während vor allem differenzierte Zellen in Apoptose gingen. Außerdem wurde der Verlust der Pluripotenz in differenzierenden ES Zellkulturen untersucht. Marker-basierte Analysen und funktionelle Tests wurden mit ES Zellen durchgeführt, die kurzfristig in vitro differenziert wurden. Es stellte sich heraus, dass nach funktionellen Gesichtspunkten die Pluripotenz bereits nach 2 Tagen Differenzierung deutlich reduziert war, beurteilt anhand der Fähigkeit Kolonien zu bilden, embryoide Körperchen (EK) zu formieren und zu kontrahierenden Herzmuskelzelltypen zu differenzieren. Im Gegensatz dazu verringerte sich die Expression von Pluripotenzmarkern erst zu späteren Zeitpunkten. Ich habe weiterhin beobachten können, dass die Wahl des Differenzierungssystems (Aggregations-EK, klonale EKs oder als adhärente Einzelzellschicht) einen Einfluss auf den Fortschritt und die Homogenität der Differenzierung hatte. Um das Ende der Pluripotenz genauer zu untersuchen, wurden differenzierte ES Zellen zurück in ES Zellkulturbedingungen gebracht. Die Ergebnisse deuten an, dass 3 Tage differenzierte ES Zellen einen Punkt überschritten haben, an dem eine Rückkehr zur Pluripotenz allein durch Kulturbedingungen noch möglich ist. Durch die Behandlung mit HDIs starben selektiv differenzierte ES Zellen. Des Weiteren war es Ziel dieser Arbeit, den Einuss von EED - einer essentiellen Untereinheit des Histon-methylierenden Polycomb repressive complex 2 (PRC2) - auf das Chromatin und die Funktion von ES Zellen hin zu analysieren. ES Zellen ohne EED wiesen neben dem bereits bekannten Verlust der Trimethylierung von Histon 3 an Lysin 27 (H3K27me3), global reduzierte H3K9me3 Levels sowie erhöhte Histonazetylierung auf. Trotz typischer ES Zell-Morphologie und normaler Expression von Pluripotenzgenen, besaßen EED knockout (KO)ES Zellen eine veränderte Organisation der Heterochromatinstruktur im Zellkern, eine verlangsamte Chromatinmobilität und Probleme bei der Differenzierung. Zusammenfassend gewähren meine Daten Einblick in die epigenetische Regulation von Stammzellen. Im Besonderen konnte ich zeigen, dass die Behandlung mit HDIs für differenzierende Knochenmarkzellen und differenzierende ES Zellen nachteilig war und zu deren selektivem Zelltod führte. Die hier durchgeführten Analysen ergaben, dass ES Zellen nach 3 Tagen Differenzierung das Ende der Pluripotenz erreicht hatten. Schließlich zeigten die Versuche mit EED KO ES Zellen, dass sie sich zwar selbst erneuerten und morphologisch identisch mit wildtypischen ES Zellen waren, jedoch Defekte bei der Differenzierung besaßen. Dies deutet darauf hin, dass EED nicht nur für undifferenzierte ES Zellen wichtig ist, sondern auch während der Differenzierung von Bedeutung ist. / Stem cells with the particular potential to self renew and to differentiate into multiple cell lineages are fascinating cell types for basic and applied research. Pluripotent embryonic stem (ES) cells are derived from the inner cell mass (ICM) of preimplantation embryos. Upon differentiation ES cells can give rise to cells of ecto-, meso- and endoderm including germ cells. In contrast, multipotent adult stem cells are more restricted in their differentiation outcomes,they differentiate into cells of their tissue of origin. For example, hematopoietic stem cells (HSCs) that reside in hemogenic tissues such as the bone marrow (BM) differentiate into hemato-/lymphoid cell lineages. Upon differentiation of stem cells not the genome, but the epigenetic regulation changes. Differentiation-associated epigenetic changes generate cell types with distinct phenotypes and functions. For stem cell-based therapies it is important to deeper understand the relation between epigenome and cellular function. In the scope of this thesis I aimed to analyze cultures of differentiating stem cells with respect to gene expression, chromatin regulation and differentiation potential. For the analysis of global histone modification levels, which represent one mechanism for epigenetic regulation, fow cytometric protocols were established that allow single cell measurements. By applying this methodology decreased histone acetylation levels were shown in differentiated ES cell populations. In contrast, comparable histone acetylation levels were observed in differentiated and undifferentiated BM cells. In addition, I investigated effects of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) on murine BM cells, comprising also HSCs. Upon TSA treatment the frequency of cells with in vitro and in vivo hematopoietic activity was increased, while lineage committed cells underwent apoptosis. Next, the loss of pluripotency was assessed in differentiating ES cell cultures. Using short-term in vitro differentiation protocols marker-based analyses and functional assays were performed.Functionally pluripotency was diminished after 2 days of differentiation as assessed by colony formation, embryoid body (EB) formation and cardiomyogenic differentiation approaches. In contrast, pluripotency marker expression was reduced at later time points. Further, the application of distinct differentiation systems (aggregation EB, clonal EB or monolayer (ML) culture) had an impact on the progression and homogeneity of differentiation cultures. To further study the end of pluripotency, differentiated ES cells were placed under ES cell culture conditions. The data suggest that 3 days differentiated ES cells had passed a point of no return and failed to regain Oct4-eGFP expression and that HDAC inhibitor treatment selectively killed differentiated ES cells. Finally, I aimed to study the effect of EED - a core subunit of the histone methylating Polycomb repressive complex 2 (PRC2) - on ES cell chromatin and function. ES cells lacking EED showed loss of histone H3 lysine 27 trimethylation (H3K27me3) accompanied by increased histone acetylation and reduced H3K9me3 levels. Despite typical ES cell morphology and pluripotency marker expression, EED knockout (KO) ES cells exhibited altered nuclear heterochromatin organization, delayed chromatin mobility and a failure in proper differentiation. Conclusively, my data provide insights into the epigenetic regulation of stem cells. Particularly, the results suggest that HDAC inhibitor treatment was detrimental for differentiated BM as well as for differentiated ES cells and that ES cells after 3 days of differentiation had lost pluripotency. Further, the data demonstrate that EED KO ES cells self renewed, exhibited morphology and pluripotency marker expression similar to wild type ES cells, but failed to differentiate. This indicates an important role of EED not only for undifferentiated but also for differentiating ES cells.

Pluripotency describes the ability of stem cells to form every cell type of the body.. Pluripotent stem cells are e.g. embryonic stem cells (ESCs), but also the so called induced pluripotent stem cells (IPS cells), that are generated by reprogramming differentiated somatic cells into a pluripotent state. Furthermore, it has been shown that spermatogonia (SG) derived from adult testes of mouse or human are pluripotent. Because of their ability to differentiate into every somatic cell type, pluripotent stem cells have a unique status in research and regenerative medicine. For the latter, they offer a valuable opportunity to replace destroyed tissues or organs. For basic research, stem cells represent a useful system to study differentiation or developmental processes that are difficult to access in the physiological situation e.g. during embryogenesis. Both applications, however, require methods that allow efficient and directed differentiation of stem cells into defined specialized cell types. This study first aims to investigate the differentiation potential of SG derived from the teleost fish medaka (Oryzias latipes). My results demonstrate that medaka SG are able to form different somatic cell types, namely adipocytes, melanocytes, osteoblasts, and neurons. This indicates that medake SG have retained a broad differentiation potential suggesting that pluripotency is not restricted to mouse and human SG but might be conserved among vertebrates. Next, I wanted to establish a differentiation method that is solely based on ectopic expression of genes known to be essential for the formation of certain somatic cell types – so called master regulators (MRs). My findings show that ectopic expression of the melanocyte-specific transcription factor mitf-m that has previously been shown to induce differentiation of medaka ESCs into pigment cells resulted in the formation of the same cell type in medaka SG. This approach could be used to generate other somatic cell types. Thus, ectopic expression of the MRs cbfa1 and mash1 in MF-SG was sufficient to induce differentiation into osteoblasts and neurons, respectively. Interestingly, these differentiation processes included the activation of genes that are expressed earlier during embryogenesis than the differentiation-inducing MR. Furthermore, my findings show that the approach of MR-induced differentiation can be transferred to mammalian stem cell systems. Ectopic expression of the neural transcription factor ngn2 was sufficient to induce efficient and rapid differentiation of neurons in mouse ESCs. This differentiation process also included the induction of genes that in vivo are activated at earlier stages that ngn2. By generating a transgenic cell line allowing induction of ectopic ngn2 expression, it was possible to obtain a relatively pure culture of functional neurons. Ngn2-induced differentiation did not require any additional signals and occurred even under pluripotency promoting conditions. Moreover, ectopic expression of ngn2 did also induce the formation of cells with neuronal morphology in IPS cells indicating that MR-induced differentiation is operative in different stem cell types. Furthermore, protein transduction of Ngn2 into mouse ESCs also resulted in a neuronal differentiation process up to the appearance of neural precursor cells. Last, my results show that MR-induced differentiation can also be used to generate other cell types than neurons from mouse ESCs. Myoblasts and macrophage-like cells were generated by ectopic expression of the MRs myoD and cebpa, respectively. Using transgenic cell lines enabling induction of MR expression it was possible to obtain mixed cultures with two different differentiation processes occurring in parallel. Altogether this study shows that ectopic expression of single genes is sufficient to induce directed differentiation of stem cells into defined cell types. The feasibility of this approach was demonstrated for different MRs and consequently different somatic cell types. Furthermore, MR induced differentiation was operative in different stem cell types from fish and mouse. Thus, one can conclude that certain genes are able to define cell fates in in vitro stem cell systems and that this cell fate defining potential appears to be a conserved feature in vertebrates. These findings therefore provide new insights in the role of MRs in cell commitment and differentiation processes. Furthermore, this study presents a new method to induce directed differentiation of stem cells that offers several advantages regarding efficiency, rapidness, and reproducibility. MR-induced differentiation therefore represents a promising tool for both stem cell research and regenerative medicine. / Pluripotenz bezeichnet die Fähigkeit einer Stammzelle, jede Zelle des Körpers zu bilden. Zu den pluripotenten Stammzellen gehören embryonale Stammzellen (ESZ), aber auch so genannte induzierte pluripotente Stammzellen (IPS Zellen), die durch Rückprogrammierung ausdifferenzierter Körperzellen in einen pluripotenten Status gewonnen werden. Außerdem wurde gezeigt, dass adulte Spermatogonien (SG) in Maus und Mensch pluripotent sind. Pluripotente Stammzellen sind von großer Wichtigkeit für Forschung und regenerative Medizin. Für letztere bieten diese Zellen aufgrund ihrer Fähigkeit, jede Körperzellen zu bilden, eine vielversprechende Möglichkeit, zerstörte Gewebe oder Organe zu ersetzen. In der Forschung stellen sie ein nützliches System dar, um Entwicklungs- und Differenzierungsprozesse zu untersuchen, die in der physiologischen Situation z.B. der Embryonalentwicklung – schwer zugänglich sind. Eine wichtige Grundlage für diese Anwendungen sind jedoch Methoden, die die effiziente und gerichtete Differenzierung von Stammzellen in einen bestimmten Zelltyp erlauben. In dieser Arbeit wird zunächst das Differenzierungspotential von SG der Fischspezies Medaka (Oryzias latipes) untersucht, um festzustellen, ob Pluripotenz von SG, die bisher nur in Maus und Mensch gezeigt wurde, auch in anderen Wirbeltieren außerhalb der Säuger erhalten ist. Meine Ergebnisse zeigen, dass Medaka-SG fähig sind verschiedene somatische Zelltypen zu bilden. Das zweite Ziel dieser Studie ist die Entwicklung einer Differenzierungsmethode, die nur auf der Expression einzelner so genannter Masterregulatoren (MR) beruht – Gene, die als essentiell für die Entwicklung bestimmter Zelltypen bekannt sind. Meine Ergebnisse zeigen, dass der Pigmentzell-spezifische Transkriptionsfaktor Mitf-M, von dem gezeigt wurde, dass er die Differenzierung von Medaka-ESZ in Pigmentzellen induzieren kann, die Bildung desselben Zelltyps in Medaka-SG induziert. Dieser Ansatz ermöglichte auch die Bildung anderer somatischer Zelltypen. So führte Überexpression der MR cbfa1 und mash1 in Medaka SG zur Differenzierung in Osteoblasten bzw. Neuronen. Interessanterweise wurde bei diesen Differenzierungsprozessen die Aktivierung von Genen beobachtet, die während der Embryonalentwicklung vor dem Differenzierung-auslösenden MR aktiviert werden. Weiterhin zeigen meine Ergebnisse, dass der Ansatz einer gerichteten Differenzierung, ausgelöst durch einzelne MR, auch auf Säuger-Stammzellen übertragen werden kann. So wurde durch Überexpression des neuronalen Genes ngn2 in murinen ESZ die effiziente und schnelle Bildung von Nervenzellen induziert, wobei auch hier die Aktivierung von Genen beobachtet wurde, deren Expression in der Embryogenese der von ngn2 vorangeht. Die Herstellung einer transgenen Zelllinie, in der die Überexpression von ngn2 aktiviert werden kann, erlaubte die Entstehung einer fast reinen Kultur funktionaler Neuronen. Der durch ngn2 ausgelöste Differenzierungsprozess war unabhängig von zusätzlichen Faktoren und lief sogar unter Bedingungen ab, die normalerweise den pluripotenten Zustand unterstützen. Außerdem führte Überexpression von ngn2 auch in IPS Zellen zur Bildung von Zellen mit neuronalem Phenotyp. Weiterhin konnte auch durch Transduktion des Ngn2-Proteins in murine ESZ neuronale Differenzierung ausgelöst werden, und zwar die Bildung neuronaler Vorläuferzellen. Zuletzt wird bewiesen, dass gerichtete Differenzierung von murinen ESZ durch einzelne MR Gene neben neuronalen Zelltypen auch die Bildung anderer somatischer Zellen erlaubt: Überexpression der Gene myoD oder cebpa induzierte die Differenzierung in Muskelzellen bzw. Macrophagen-ähnliche Zellen. Unter Verwendung transgener Zelllinien, die die Aktivierung jeweils eines MRs erlauben, war es möglich, gemischte Kulturen zu erhalten, in denen zwei verschiedene Differenzierungsprozesse parallel abliefen. Diese Studie zeigt, dass die Überexpression einzelner Gene ausreichend ist, um gerichtete Differenzierungsprozesse in einen bestimmten Zelltyp auszulösen. Die erfolgreiche Durchführung dieses Ansatzes wird nicht nur mit verschiedenen Genen und somit verschiedenen resultierenden Zelltypen nachgewiesen, sondern auch in verschiedenen Stammzelltypen aus Fisch und Maus. Dies erlaubt die Schlussfolgerung, dass bestimmte Gene in vitro das Schicksal von Stammzellen festlegen können und dass diese Fähigkeit eine konservierte Eigenschaft in Wirbeltieren zu sein scheint. Somit präsentiert diese Arbeit neuen Erkenntnisse über die Rolle von MR bei der Festlegung von Zellidentitäten und in Differenzierungsprozessen. Weiterhin wird eine neue Methode zur Induktion gerichteter Differenzierung in Stammzellen aufgezeigt, die mehrere Vorteile in Bezug auf Effizienz, Geschwindigkeit und Reproduzierbarkeit hat. Auslösung von Differenzierung durch MR Gene bietet somit einen neuen vielversprechenden Ansatz mit potentieller Anwendung sowohl in Stammzellforschung, als auch in regenerativer Medizin.

Polycomb group (PcG) proteins are chromatin modifiers involved in heritable gene repression. Two main PcG complexes have been characterized: Polycomb repressive complex (PRC) 2 is involved in the initiation of gene silencing, whereas PRC1 participates in the stable maintenance of gene repression. Pcgf4 (Polycomb group protein, Bmi1) is one of the most studied PRC1 members with essential functions for embryonic development and adult stem cell self renewal. In embryonic stem cells (ES cells), Pcgf4 is poorly expressed while its paralogs (Pcgf1, Pcgf2, Pcgf3, Pcgf5 and Pcgf6) are expressed at higher levels. The relevance of the Pcgf paralog Pcgf6 for the maintenance of ESC pluripotency has not been addressed so far. My analyses revealed that Pcgf6 was the most expressed Pcgf paralog in undifferentiated ES cells. When ES cells differentiated, gene expression of Pcgf6 strongly declined. To investigate the functions of Pcgf6 in ES cells, we established a doxycycline (dox) inducible shRNA-targeted knockdown system according to publications by Seibler et al. (Seibler et al. 2005; Seibler et al. 2007). Following dox-induced knockdown (KD) of Pcgf6, we observed decreased ES cell colony formation. In parallel, gene expression of pluripotency markers Oct4, Nanog and Sox2 was reduced upon dox-treatment, wheras the expression of mesoderm genes such as T (Brachyury) were up-regulated. Further, microarray analysis revealed de-repression of several spermatogenesis-specic genes upon Pcgf6-KD, suggesting that Pcgf6 may play a role during spermatogenesis. Upon in vitro differentiation, Pcgf6-KD ES cells showed increased hemangioblast formation, paralleled by increased hematopoietic development. In summary, results of this study suggest that Pcgf6 is involved in maintaining ES cell identity by repressing lineage-specific gene expression in undifferentiated ES cells. / Polycomb Gruppe (PcG) Proteine sind Chromatin-Modifikatoren, die an der vererbbaren Genrepression beteiligt sind. Primär wurden bisher zwei PcG-Komplexe charakterisiert: Polycomb-repressiv-Komplex (PRC) 2, der die ersten Schritte des Gen-Silencings übernimmt, und PRC1, der an der stabilen Aufrechterhaltung der Genrepression beteiligt ist. Pcgf4 (Bmi1) ist das am besten untersuchte PRC1-Mitglied. Pcgf4 hat wichtige Funktionen in der embryonalen Entwicklung und in der Selbst-Erneuerung adulter Stammzellen. In embryonalen Stammzellen (ES-Zellen) wird Pcgf4 kaum exprimiert, während seine Paraloge (Pcgf1, Pcgf2, Pcgf3, Pcgf5 und Pcgf6) höher exprimiert sind. Die Bedeutung des Pcgf-Paralogs Pcgf6 für die Aufrechterhaltung der Pluripotenz von ES-Zellen wurde bislang nicht untersucht. Meine Analysen zeigten, dass Pcgf6 der am meisten exprimierter Pcgf-Paralog in undifferenzierten ES-Zellen war. Während der Differenzierung von ES-Zellen wurde die Expression von Pcgf6 stark reduziert. Um die Funktionen von Pcgf6 in ES-Zellen zu untersuchen, habe ich ein Doxycyclin (dox)-induzierbares shRNA-Expressionssystem für den gezielten Knockdown (KD) von Pcgf6 nach Seibler et al. (Seibler et al. 2005; Seibler et al. 2007) etabliert. Nach dox-induziertem KD von Pcgf6 beobachtete ich eine Verringerung der ES-Zell-Kolonie-Bildung. Die Expression der Pluripotenzmarker Oct4, Nanog und Sox2 war nach Dox-Behandlung reduziert, während die Expression mesodermaler Gene, wie z.B. T (Brachyury), hochreguliert wurden. Außerdem zeigten Microarray-Analysen eine De-Repression Spermatogenese-spezifischer Gene nach KD von Pcgf6, was darauf hindeutete, dass Pcgf6 eine Rolle in der Spermatogenese spielen könnte. In der in-vitro- Differenzierung zeigten Pcgf6-KD-ES-Zellen, neben einer erhöhten Bildung von Hämangioblasten, mehr hämatopoetische Vorläufer. Zusammenfassend zeigten die Daten dieser Studie, dass das Pcgf-Paralog Pcgf6 an der Aufrechterhaltung der ES-Zell-Identität durch Unterdrücken lineage-spezifischer Geneexpression in undifferenzierten ES-Zellen beteiligt ist.

Regeneration is a process that renews damaged or lost cells, tissues, or even of entire body structures, and is a phenomenon which is widespread in the animal kingdom. Urodeles such as newts and salamanders have a remarkable regeneration ability. They can regenerate organs such as gills, lower jaws, retina, appendages like fore- and hind limbs, and also the tail including the spinal cord. The regeneration process requires the use of resident stem cells or somatic cells, which have to be reprogrammed. In both cases the reprogrammed cells are less differentiated, meaning the cell would have the ability to form any kind of fetal or adult cell which rose from the three different germ layers, the ectoderm, mesoderm and endoderm. Artificial reprogramming of differentiated mammalian somatic cell had been reported previously. It was shown that four pluripotency factors, OCT4 (also called POU5f1), SOX2, c-MYC and KLF4 are sufficient to generate an induced pluripotent stem (iPS) cell. It has been shown that some of these factors are also involved in regenerating processes. In newt limb and lens tissue, Sox2, c-Myc and Klf4 mRNA levels were upregulated in the beginning of blastema formation when compared to non-amputated tissue. Oct4 mRNA however, was not detected. During xenopus tail regeneration, Sox2 and c-Myc were expressed, while the xenopus Pou homologs Pou25, Pou60, Pou79, Pou91 were not detected. In regenerating zebrafish fin tissue, Sox2, Pou2, c-Myc and Klf4 mRNA were not upregulated. The mammalian transcription factor OCT4, a class V POU protein, is responsible in maintaining pluripotency in gastrula stage embryos. It was reported that mouse OCT4 is also expressed in the caudal node of embryos having 16 somites. It is further known that progenitors exist in mouse tailbud, which give rise to neural and mesodermal cell lineage. This suggests that the OCT4 expressing cells in caudal node might be a stem cell reservoir. Oct4 was detected in axolotl during embryonic development, and prior to my work we found Oct4 when screening the axolotl blastema cDNA library. In addition, we also identified Pou2, another class V POU gene. Phylogenetic analysis showed a clear distinction of both genes in the axolotl. We determined the mRNA pattern of Pou2 during embryogenesis and compared it to Oct4 mRNA and protein. Both genes are expressed in the primordial germ cells and the pluripotent animal cap region of the embryo. Apart from this similarity, both genes have a different expression pattern in the embryo. We are interested in the involvement of OCT4, POU2, as well as the transcription factor SOX2 in regenerating axolotl spinal cord. We asked whether the cellular pluripotent character conferred by POU factors is limited to mammals or if it is an ancient characteristic of lower vertebrates. To answer the question we performed in vitro and in vivo studies. Hence this thesis is separated into two chapter. By in vitro studies we investigated the pluripotent PouV orthologs from different species. Therefore, we performed reprogramming experiments using mouse or human fibroblasts and transduced them with axolotl Oct4 or Pou2, in combination with human or axolotl Sox2, c-Myc and/or Klf4. The generated iPS cells with the different sets of factors had similar endogenous pluripotency gene expression profiles to embryonic stem cells. Further, iPS cells expressed the pluripotency markers like OCT4, NANOG, SSEA4, TRA1-60 and TRA1-81. Another evaluation of the iPS cells was the formation of embryoid bodies. Immunouorescence staining showed that tissue from all three germ layers was formed after induction. We observed a positive staining for the endoderm marker !-FEROPROTEIN, the mesoderm marker !-SMOOTH MUSCLE ACTIN and the ectoderm marker \"III TUBULIN in the generated cells. This indicated that the iPS cells generated using axolotl Oct4 and Sox2 in combination with mammalian Klf4 and with or without c-Myc, as well as iPS cell generated with axolotl Pou2 and mammalian Sox2 and Klf4 and with or without c-Myc have a pluripotent potential. In addition, the axolotl factors are able to form heterodimers with the mammalian proteins. Furthermore, we compared the reprogramming ability with POU factors from mouse, human, zebrash, medaka and xenopus. We showed that xenopus Pou91, as the only non-mammalian example, is nearly as efficient as mouse and human Oct4 cDNAs in inducing GFP expressing cells. Also axolotl Pou2, axolotl Oct4 and medaka Pou2 showed reprogramming character however at a much lower efficiency. In contrast, zebrash Pou2 is not able to establish iPS cells. This indicates that a reprogramming ability to a pluripotent cell state is an ancient trait of Pou2 and Oct4 homologs. By in vivo studies we investigated the role of Oct4, Pou2 and Sox2 gene expression in regenerating spinal cord tissue. Performed in situ hybridizations and antibody staining studies in the regenerating spinal cord showed that Oct4, Pou2 and Sox2 were expressed during spinal cord regeneration. Knockdown experiments in regenerating spinal cord using morpholino showed that Pou2-morpholino does not have an effect. In contrast, SOX2 was required for spinal cord regeneration but to a lesser extent, than OCT4, which decreased the regenerated length signicantly compared to control. Even though, with Sox2-morpholino we did not observe the phenotype as a significantly shorter regenerated spinal cord, about 45% of SOX2 knocked down cells were not cycling and proliferating anymore. This indicates that axolotl SOX2 has an effect in regeneration. Therefore we wanted to know whether spinal cord cells would also have a pluripotent character in vivo and form other tissue types. Regenerating cells of the spinal cord are only able to form the same cell type and thus they keep their cell memory. However, when we performed transplantations of OCT4/SOX2 expressing spinal cord cells into somite stage embryos, we could show the formation of muscle cells. This shows that the spinal cord cells have the potential to change their fate in an embryonic context, where the normal environment of spinal cord has changed. However, our data do not indicate whether muscle is formed directly from the spinal cord or whether spinal cord cells fuse to developmental myoblasts, a cell type of embryonic progenitors, which give rise to muscle cells. To clearly state whether regenerating OCT4/SOX2 expressing spinal cord cells are pluripotent we have to perform OCT4 knock down in spinal cord and transplant these less proliferating cells into embryos, observing their cell fate.

Regeneration is a process that renews damaged or lost cells, tissues, or even of entire body structures, and is a phenomenon which is widespread in the animal kingdom. Urodeles such as newts and salamanders have a remarkable regeneration ability. They can regenerate organs such as gills, lower jaws, retina, appendages like fore- and hind limbs, and also the tail including the spinal cord. The regeneration process requires the use of resident stem cells or somatic cells, which have to be reprogrammed. In both cases the reprogrammed cells are less differentiated, meaning the cell would have the ability to form any kind of fetal or adult cell which rose from the three different germ layers, the ectoderm, mesoderm and endoderm. Artificial reprogramming of differentiated mammalian somatic cell had been reported previously. It was shown that four pluripotency factors, OCT4 (also called POU5f1), SOX2, c-MYC and KLF4 are sufficient to generate an induced pluripotent stem (iPS) cell. It has been shown that some of these factors are also involved in regenerating processes. In newt limb and lens tissue, Sox2, c-Myc and Klf4 mRNA levels were upregulated in the beginning of blastema formation when compared to non-amputated tissue. Oct4 mRNA however, was not detected. During xenopus tail regeneration, Sox2 and c-Myc were expressed, while the xenopus Pou homologs Pou25, Pou60, Pou79, Pou91 were not detected. In regenerating zebrafish fin tissue, Sox2, Pou2, c-Myc and Klf4 mRNA were not upregulated. The mammalian transcription factor OCT4, a class V POU protein, is responsible in maintaining pluripotency in gastrula stage embryos. It was reported that mouse OCT4 is also expressed in the caudal node of embryos having 16 somites. It is further known that progenitors exist in mouse tailbud, which give rise to neural and mesodermal cell lineage. This suggests that the OCT4 expressing cells in caudal node might be a stem cell reservoir. Oct4 was detected in axolotl during embryonic development, and prior to my work we found Oct4 when screening the axolotl blastema cDNA library. In addition, we also identified Pou2, another class V POU gene. Phylogenetic analysis showed a clear distinction of both genes in the axolotl. We determined the mRNA pattern of Pou2 during embryogenesis and compared it to Oct4 mRNA and protein. Both genes are expressed in the primordial germ cells and the pluripotent animal cap region of the embryo. Apart from this similarity, both genes have a different expression pattern in the embryo. We are interested in the involvement of OCT4, POU2, as well as the transcription factor SOX2 in regenerating axolotl spinal cord. We asked whether the cellular pluripotent character conferred by POU factors is limited to mammals or if it is an ancient characteristic of lower vertebrates. To answer the question we performed in vitro and in vivo studies. Hence this thesis is separated into two chapter. By in vitro studies we investigated the pluripotent PouV orthologs from different species. Therefore, we performed reprogramming experiments using mouse or human fibroblasts and transduced them with axolotl Oct4 or Pou2, in combination with human or axolotl Sox2, c-Myc and/or Klf4. The generated iPS cells with the different sets of factors had similar endogenous pluripotency gene expression profiles to embryonic stem cells. Further, iPS cells expressed the pluripotency markers like OCT4, NANOG, SSEA4, TRA1-60 and TRA1-81. Another evaluation of the iPS cells was the formation of embryoid bodies. Immunouorescence staining showed that tissue from all three germ layers was formed after induction. We observed a positive staining for the endoderm marker !-FEROPROTEIN, the mesoderm marker !-SMOOTH MUSCLE ACTIN and the ectoderm marker \"III TUBULIN in the generated cells. This indicated that the iPS cells generated using axolotl Oct4 and Sox2 in combination with mammalian Klf4 and with or without c-Myc, as well as iPS cell generated with axolotl Pou2 and mammalian Sox2 and Klf4 and with or without c-Myc have a pluripotent potential. In addition, the axolotl factors are able to form heterodimers with the mammalian proteins. Furthermore, we compared the reprogramming ability with POU factors from mouse, human, zebrash, medaka and xenopus. We showed that xenopus Pou91, as the only non-mammalian example, is nearly as efficient as mouse and human Oct4 cDNAs in inducing GFP expressing cells. Also axolotl Pou2, axolotl Oct4 and medaka Pou2 showed reprogramming character however at a much lower efficiency. In contrast, zebrash Pou2 is not able to establish iPS cells. This indicates that a reprogramming ability to a pluripotent cell state is an ancient trait of Pou2 and Oct4 homologs. By in vivo studies we investigated the role of Oct4, Pou2 and Sox2 gene expression in regenerating spinal cord tissue. Performed in situ hybridizations and antibody staining studies in the regenerating spinal cord showed that Oct4, Pou2 and Sox2 were expressed during spinal cord regeneration. Knockdown experiments in regenerating spinal cord using morpholino showed that Pou2-morpholino does not have an effect. In contrast, SOX2 was required for spinal cord regeneration but to a lesser extent, than OCT4, which decreased the regenerated length signicantly compared to control. Even though, with Sox2-morpholino we did not observe the phenotype as a significantly shorter regenerated spinal cord, about 45% of SOX2 knocked down cells were not cycling and proliferating anymore. This indicates that axolotl SOX2 has an effect in regeneration. Therefore we wanted to know whether spinal cord cells would also have a pluripotent character in vivo and form other tissue types. Regenerating cells of the spinal cord are only able to form the same cell type and thus they keep their cell memory. However, when we performed transplantations of OCT4/SOX2 expressing spinal cord cells into somite stage embryos, we could show the formation of muscle cells. This shows that the spinal cord cells have the potential to change their fate in an embryonic context, where the normal environment of spinal cord has changed. However, our data do not indicate whether muscle is formed directly from the spinal cord or whether spinal cord cells fuse to developmental myoblasts, a cell type of embryonic progenitors, which give rise to muscle cells. To clearly state whether regenerating OCT4/SOX2 expressing spinal cord cells are pluripotent we have to perform OCT4 knock down in spinal cord and transplant these less proliferating cells into embryos, observing their cell fate.

No description available.

No description available.

Unbegrentzte Selbsterneuerungkapazität und Pluripotenz sind charakteristische Merkmale von embryonalen Stammzellen (ES-Zellen). Dennoch sind die molekularen und zellulären Mechanismen, die für das Schicksal der ES-Zellen zuständig sind, noch nicht genau definiert. Um regulierende Faktoren des undifferenzierten Zustands von ES-Zellen zu identifizieren, wurden undifferenzierte ES Zellen, "Embryoid Bodies", spontan differenzierte und mit Retinsäure differenzierte ES Zellen mittels Microarray-Analysen verglichen. Neben bereits etablierten Pluripotenz-Markern, wurde Sprouty4 als eines der am stärksten degerulierten Transkripte unter diesen Bedingungen identifiziert. Sprouty4 ist als Inhibitor des ERK (Extracellular signal-regulated protein kinase)-Signalweges bekannt, aber seine Rolle in ES-Zellen wurde noch nicht definiert. Mittels Genexpression und Western BlotAnalysen konnte gezeigt werden, dass Sprouty4 in undifferenzierten ES-Zellen stark exprimiert ist und im Verlauf der Differenzierung schnell herunterreguliert wird. In vivo war Sprouty4 auf die innere Zellmasse (ICM) der Mausblastozyste beschränkt. Außerdem wurde gezeigt, dass der Sprouty4 Promotor durch direkte Bindung der PluripotenzMarkern Nanog, Klf4 und Stat3 reguliert wird. ES-Zellen, die Sprouty4 konstitutiv exprimieren, waren resistent gegen Differenzierung durch Zugabe von Retinsäure oder Bildung von Embryoid Bodies. Hingegen führte die Expression einer dominant-negativen Mutante von Sprouty4 zu einer erhörten Sensitivierung von ES-Zellen gegenüber der Differenzierung und zur Bildung extraembryonaler Gewebe begleitet von Endoreduplikation. Zusammenfassend konnten unsere Ergebnisse zeigen, dass die enge Regulation des ERK-Signalweges und warscheinlich anderer Signalwege durch Sprouty4 notwendig ist, um die Balance zwichen Pluripotenz und Differenzierung embryonaler Stammzellen zu kontrollieren. / A hallmark feature of embryonic stem (ES) cells is the ability to self-renew indefinitely while maintaining pluripotency. However, the molecular and cellular mechanisms underlying ES cell fate are poorly understood. To identify signaling pathway molecules that maintain the uncommitted state of ES cells, a microarray analysis was performed comparing undifferentiated ES cells, mature embryoid bodies, spontaneously differentiated and retinoic acid-induced differentiated ES cells. Among several well-validated pluripotency markers, Sprouty4 was identified as one of the most highly deregulated transcripts under these conditions. Sprouty4 is known as an inhibitor of the extracellular signal-regulated protein kinase (ERK/MAPK) pathway however its role in ES cells has not yet been defined. Gene expression and western-blot analyses have shown that Sprouty4 is highly expressed in ES cells and strongly downregulated upon differentiation whilst in vivo, Sprouty4 is confined to the founder population of ES cells, the inner cell mass of mouse blastocysts. Moreover, the Sprouty4 promoter was found to be regulated via the direct binding of the intrinsic pluripotency-associated factors Nanog, Klf4 and Stat3. ES cells engineered to constitutively express a wild-type version of Sprouty4 were found to be resistant to differentiation induced by retinoic acid or embryoid bodies formation. Conversely, expression of a dominant negative Sprouty4 mutant activating the ERK/MAPK pathway in a sustained manner sensitized ES cells to differentiation and triggered endoreduplication leading to the formation of extraembryonic tissue. Taken together, these results highlight the essential role of Sprouty4 in the tight regulation of the ERK/MAPK pathway- and probably others- for the balance between pluripotency and lineage commitment in mouse embryonic stem cells.