Tesis sobre el tema "Leukemia inhibitory factor"

Addendum attached to back facing leaves. Includes bibliographical references (leaves 172-199). The findings establish leukemia inhibitory factor, and possibly oncostatin M, as new members of a small but growing class of cytokines produced in an intracellularly active form and also suggest that the production of alternate transcripts and intercellularly-retained proteins may be a common and important feature of cytokines of the IL-6 and other families.

Background: Leukemia inhibitory factor (LIF) is an interleukin 6 class cytokine that inhibits cell differentiation during cell development. LIF is widely used as an important component in stem cell culture medium for cell therapy and tissue engineering applications, to inhibit spontaneous cell differentiation. However, commercially available recombinant LIF proteins are expensive, unstable, and prone to degradation in prolonged tissue culture, which has limited its applications. Purpose: We aim to develop a lab-scale method, using a yeast cell, namely, P. pastoris, to produce recombinant hLIF (rhLIF), with novel recombinant protein expression techniques. Methods: We first designed the structure of plasmid and generated the plasmid using the service of an external supplier. The plasmid was amplified in E. coli using a heat shock method. The amplified plasmid was then linearized and transcribed in the P. pastoris. Methanol was used in the medium to induce the expression of rhLIF in P. pastoris. The resultant rhLIF was tested verified by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) Results: The plasmids were successfully amplified in E. coli. The generation of rhLIF in P. pastoris was verified by the SDS-PAGE results. To our knowledge, it is the first study to express rhLIF using lower eukaryotic yeast. Future direction: In the future studies, we will study the glycosylation sites of rhLIF using liquid chromatography–mass spectrometry (LC–MS). In addition, the function and stability of the rhLIF will be compared with commercial products.

Previous publications from this laboratory demonstrated that administration of leukemia inhibitory factor (LIF) (125 µg/kg) to young, male Sprague-Dawley rats at 6, 24, and 48 h after middle cerebral artery occlusion (MCAO) reduced infract volume, improved sensimotor skills, and alleviated damage to white matter at 72 h after the injury. In vitro studies using cultured oligodendrocytes (OLs) showed that LIF (200 ng/ml) also protects against 24 h of oxygen-glucose deprivation through activation of Akt signaling and upregulation of the antioxidant enzymes peroxiredoxin IV and metallothionein III. Other groups have demonstrated that LIF reduces neurodegeneration in animal models of disease, but the neuroprotective mechanisms of LIF during permanent ischemia have not yet been examined. The overall hypothesis to be tested in this project is whether LIF exerts similar protective mechanisms against neurons during ischemia through increased antioxidant enzyme expression in neurons. In the first set of experiments, superoxide dismutase (SOD) activity was significantly increased in the ipsilateral hemisphere of LIF-treated rats compared to rats that received PBS treatment at 72 h after MCAO. Western blot and immunohistochemical analysis revealed that SOD3 was upregulated in brain tissue and induced specifically in cortical neurons tissue at this time point. Neurons that expressed high levels of SOD3 at 72 h after MCAO also showed high levels of phosphorylated Akt (Ser473). LIF (200 ng/ml) reduced necrotic and apoptotic cell death against 24 h of OGD as measured by lactate dehydrogenase (LDH) release and caspase-3 activation. Quantitative real-time PCR analysis showed that LIF treatment upregulated SOD3 gene expression in vitro during OGD. Treatment with 10 µM Akt Inhibitor IV and transfection with SOD3 siRNA counteracted the neuroprotective effects of LIF in vitro, showing that upregulation of SOD3 and activation of Akt signaling are necessary for LIF-mediated neuroprotection. Several transcription factors that regulated Akt-inducible genes were previously identified by this lab, including myeloid zinc finger-1 (MZF-1) and specificity protein-1 (Sp1). The goal of the second set of experiments was to determine whether LIF exerted protective actions through MZF-1 and Sp1. According to analysis with Genomatix, MZF-1 and Sp1 have multiple binding sites in the promoter for the rat SOD3 gene. Western blot analysis showed that there was a trend towards increased MZF-1 protein expression in the brains of LIF-treated rats that approached significance. Immunohistochemical analysis and quantitative real-time PCR showed a significant in vitro upregulation in MZF-1 expression among LIF-treated neurons compared to PBS-treated neurons. Sp1 gene expression was not changed by LIF treatment, but there was a trend towards increased protein expression. In addition, there was a significant correlation between Sp1 and MZF-1 among brain samples from LIF-treated rats but not PBS-treated or sham rats at 72 h after MCAO. Immunohistochemical analysis revealed that Sp1 and MZF-1 co-localized with neuronal nuclei and SOD3 at 72 h after MCAO. Neurons that were transfected with MZF-1 or Sp1 siRNA following isolation did not show a significant decrease in LDH release after 24 h OGD that was observed among neurons transfected with scrambled siRNA. These data demonstrate that Sp1 and MZF-1 are involved with the neuroprotective signaling of LIF under ischemia. This laboratory has demonstrated that LIF activates transcription of protective genes and increases the activity of transcription factors through modulation of intracellular signaling. However, the upstream signaling mechanisms of LIF during ischemia had not previously been investigated. Previous investigators found that the LIF-specific subunit of the heterodimeric LIF receptor (LIFR) is induced by CNS injury. Western blot analysis was used to determine whether LIFR was induced in the brain and the spleen, which plays a role in the peripheral immune response, after MCAO. According to these results, LIF treatment significantly upregulates LIF in the brain compared to PBS treatment or sham injury at 72 h after MCAO. Genomatix analysis of the LIFR promoter region revealed a binding site for Sp1, which is one of the transcription factors responsible for neuroprotection by LIF. At this same time point, splenic LIFR expression is significantly reduced after MCAO compared to sham injury. LIF treatment did not significantly increase LIFR expression, but did significantly increase spleen size compared to PBS treatment at 72 h after MCAO. Although there was a trend towards increased LIFR expression in the spleen from 24 h to 72 h after MCAO, this increase was not statistically significant. However, there was a significant positive correlation between spleen weight and LIFR expression among rats euthanized 24-72 h after MCAO/sham injury. In addition, there was a significant negative correlation between LIFR expression in the brain and the spleen weight, thus showing that LIFR is upregulated following the splenic response. According to findings from other groups, JAK1 has been shown to associate with the heterodimeric LIF receptor (LIFR/gp130) and directly activate PI3K/Akt signaling. To test whether JAK1 contributes neuroprotection during ischemia, cultured neurons were treated with several concentrations (2.5-50 nM) of GLPG0634, a JAK1-specific inhibitor prior to 24 h of OGD. With the exception of the 2.5 nM concentration, all concentrations of GLPG0634 significantly decreased LDH release compared to DMSO treatment, with the 5 nM concentration having the most potent effect on reducing cytotoxicity. However, the 5 nM concentration had no significant did not significantly reduce LDH release compared to DMSO treatment under 24 h of normoxic conditions. These results indicate that JAK1 activity is primarily detrimental to neurons during ischemia. Although it is possible that LIF signaling activates JAK1, it is unlikely that JAK1 is responsible for LIF-mediated neuroprotection during ischemia. The results of these experiments allowed us to determine several molecular mechanisms for LIF-mediated neuroprotection. LIF, which binds to its heterodimeric receptor, activates Akt signaling during ischemia. The transcription factors Sp1 and MZF-1, which are located downstream of Akt, bind to the promoter of the SOD3 gene. In addition, Sp1 also regulates the LIFR gene. SOD3 upregulation increases total SOD activity, which decreases apoptotic and necrotic cell death during apoptosis. Due to its ability to promote antioxidant expression and survival signaling in multiple neural cell types, LIF shows promise as a novel treatment for permanent focal cerebral ischemia.

Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2004.
Includes bibliographical references (leaves 134-172). Also available in electronic version. Access restricted to campus users.

Für medizinische Anwendungen sind Stammzellen aufgrund ihrer Eigenschaften (Selbsterneuerung, hohe Proliferationsrate und Differenzierungsmöglichkeit in verschiedene Zelltypen) beispielsweise in den Bereichen des regenerativen Gewebeersatzes und der Zelltherapie sehr interessant. In vivo umgibt die Stammzellen eine definierte Mikroumgebung, die sie unterstützt sich zu teilen, ihren undifferenzierten Status aufrecht zu erhalten und Tochterzellen für das Wachstum, die routinemäßige Erneuerung oder den Ersatz von Gewebe zu produzieren. Diese Mikroumgebungen werden als Stammzellnischen bezeichnet. Für die Kultivierung von Stammzellen in vitro muss die in vivo-Situation möglichst getreu nachgestaltet werden. Ziel der Forschung ist die Schaffung einer künstlichen Umgebung, die sowohl die funktionellen Eigenschaften einer Nische besitzt als auch frei von Risiken xenogener Pathogene oder Gewebeunverträglichkeiten für die Anwendung am humanen Organismus eingesetzt werden kann. Einen Ansatz dafür bietet beispielsweise die Kopplung von Faktoren, die für den Erhalt der Stammzelleigenschaften notwendig sind, an synthetische Oberflächen. Ausgehend vom Bedarf an Kultur- oder Modellsystemen für die Expansion von (embryonalen) Stammzellen sollte in dieser Arbeit analysiert werden, ob alternierende Maleinsäureanhydrid (MA)-Copolymere ein geeignetes Trägersystem für die biofunktionelle kovalente Immobilisierung spezifischer Zytokine sind und dadurch unter anderem als künstliche Stammzellnische Anwendung finden können. MA-Copolymere eignen sich aufgrund ihrer spontanen Reaktion mit Aminogruppen für die Immobilisierung von Proteinen. Das Zytokin LIF (Leukemia inhibitory factor) existiert in vivo auch in immobilisierter Form und ist in embryonalen Mausstammzellen (mESC) allein in der Lage, das Stammzellpotential dieser Zellen zu erhalten. Aus diesem Grund ist LIF für die Analyse der Aufgabenstellung geeignet. Nach der Charakterisierung LIF-modifizierter Oberflächen wurde die biologische Verfügbarkeit des kovalent immobilisierten Zytokins mit Hilfe von LIF-sensitiven Fibroblasten und mESC der Linie R1 überprüft. Anschließend wurde im Mausmodell in vivo der Erhalt der Pluripotenz der mESC durch immobilisiertes LIF analysiert. Dafür standen die Oberflächen Poly(ethylen-alt-maleinsäureanhydrid) (PEMA) und Poly(octadecen-alt-maleinsäureanhydrid) (POMA) jeweils ohne und mit Polyethylenglykol (PEG7)-Modifizierung zur Verfügung, an die LIF kovalent gekoppelt wurde. Zusätzlich wurde LIF physisorptiv an einer Kollagen-Fibronektin-Matrix über hydrolysiertem POMA immobilisiert. Mit Hilfe von radioaktiv markiertem LIF konnte gezeigt werden, dass die Gesamtbeladungsmenge mit Zytokin von den Eigenschaften der MA-modifizierten Träger abhing. Auf PEMA konnten mit steigenden Immobilisierungskonzentrationen höhere Belegungsdichten an der Oberfläche erreicht werden, die im analysierten Bereich eine lineare Abhängigkeit zeigten. Aufgrund der starken Quellung in wässrigen Lösungen war eine Einlagerung von LIF-Molekülen in die Polymerschicht möglich und führte bei hohen Immobilisierungskonzentrationen auch nach 3 Tagen Inkubation mit proteinhaltigem Medium noch zur Verdrängung nicht kovalent gebundener Zytokinmoleküle aus PEMA-Oberflächen. Obwohl ein Teil des LIF in die Polymerschicht eindrang, war der Großteil der Moleküle für einen spezifischen Antikörper zugänglich. Hydrophobe Oberflächen mit POMA konnten bei hohen Immobilisierungskonzentrationen weniger LIF binden und zeigten Sättigungsverhalten der Oberflächen bei einer Belegungsdichte von 178 ng/cm^2 LIF. Eine Freisetzung von LIF nach mehr als 3 Tagen konnte nicht beobachtet werden. Gleichzeitig war hier aufgrund der hydrophoben Polymerseitenketten die Antikörperzugänglichkeit deutlich reduziert. Wegen des geringen Quellungsverhaltens von POMA in wässrigen Lösungen konnte eine Einlagerung des immobilisierten Zytokins in die Polymerschicht aber ausgeschlossen werden. Die kovalente LIF-Immobilisierung über PEG7-Spacer führte im Vergleich zu den nicht PEG-modifizierten Oberflächen PEMA und POMA zu jeweils geringeren Belegungsdichten, ohne dabei den Charakter der Abhängigkeit von der Immobilisierungskonzentration zu verändern (linear für PEMA+PEG7, Sättigung für POMA+PEG7). Die schlechte Antikörperzugänglichkeit von immobilisiertem LIF auf POMA konnte durch die Einführung des PEG7-Spacers deutlich verbessert werden und erreichte einen Wert ähnlich dem der hydrophilen PEMA-Oberflächen. Kovalent immobilisiertes LIF zeigte auf den vier MA-Oberflächen homogene und definiert einstellbare Belegungsdichten auf den einzelnen Proben. Die physisorptive Immobilisierung von LIF an extrazelluläre Matrixkomponenten auf hydrolysiertem POMA führte zu inhomogenen und bereits bei geringen Immobilisierungskonzentrationen instabilen Belegungsdichten. Die Einstellung definierter Belegungsdichten und die homogene Verfügbarkeit des Zytokins sind für die spätere Anwendung bei der Kultivierung wichtig, da so allen Zellen die gleiche definierte Zytokindosis unabhängig von der Oberflächencharakteristik präsentiert wird und Populationsunterschiede vermieden werden. LIF-sensitive Mausfibroblasten der Linie NIH3T3 reagierten auf immobilisiertes LIF mit der Aktivierung des Signalwegproteins STAT3. Durch den direkten Vergleich von STAT3-Aktivierungsprofilen nach Stimulation mit gelöstem oder immobilisiertem LIF konnte gezeigt werden, dass durch beide Präsentationsformen innerhalb der ersten 15 Minuten nach Stimulationsbeginn eine starke Aktivierung von STAT3 erfolgt, die anschließend wieder abklingt. Die Profile beider Präsentationsformen unterschieden sich in ihren Intensitäten nur bei der starken STAT3-Aktivierung. Dabei ergaben sich bei gelöstem LIF aufgrund der größeren Kontaktfläche mit Zytokin (gesamte Zelloberfläche) etwas stärkere Aktivierungen. Durch die sehr ähnlichen Aktivierungsprofile konnte nachgewiesen werden, dass das Zytokin LIF für Zellen zugänglich an MA-Copolymere mit und ohne Spacer-Modifizierung immobilisiert werden kann. Dabei lag ein Teil der Moleküle in einer Konformation und Orientierung gebunden vor, die die Funktionalität des Zytokins erhalten konnten. Zwischen den Oberflächen mit kovalenter LIF-Immobilisierung konnten keine wesentlichen Unterschiede in der STAT3-Aktivierung festgestellt werden. LIF war an all diesen Oberflächen für die LIF-sensitiven NIH3T3 Mausfibroblasten biologisch verfügbar. LIF-abhängige embryonale Mausstammzellen (mESC) reagierten nach 72 Stunden LIF-Stimulation mit der Aktivierung von STAT3. Bei Belegungsdichten ab 8 ng/cm^2 kovalent immobilisiertem LIF auf POMA mit und ohne PEG7-Spacer konnten ähnliche Aktivierungen wie durch die Stimulation mit gelöstem LIF festgestellt werden. Dies bestätigte die biofunktionelle LIF-Immobilisierung. Zwischen den POMA-Oberflächen mit und ohne PEG7 war dabei kein deutlicher Unterschied erkennbar. Eine reduzierte Zugänglichkeit des Antikörpers auf POMA beeinflusste demnach die biologische Verfügbarkeit des Zytokins für die mESC nicht. Der Erhalt des Stammzellpotentials durch kovalent an POMA gebundenes LIF konnte in vitro durch die Präsenz von Oct4 im Zellkern der mESC nachgewiesen werden. Durch die instabile Immobilisierung bei physisorptiver Assoziation des Zytokins an Matrixkomponenten über hydrolysiertem POMA reduzierte sich der Erhalt des Stammzellpotentials auf diesen Oberflächen stark. Kovalent immobilisiertes LIF dagegen konnte auch während der Kultur über mehrere Passagen hinweg die Pluripotenz der murinen ESC erhalten. Nach der Fusion mit Blastozysten beteiligten sich diese kultivierten Zellen in vivo erfolgreich an der Bildung von Chimären. Dabei konnten keine Unterschiede der Chimärenhäufigkeit zwischen der Kultivierung der mESC mit gelöstem oder kovalent an POMA immobilisiertem LIF festgestellt werden. Kovalent an MA-Copolymere immobilisiertes LIF ist demnach in der Lage, gelöstes LIF vollständig zu ersetzen und über mehrere Passagen hinweg allein das Stammzellpotential von mESC zu erhalten. Die Experimente zeigten, dass sich MA-Copolymere für die funktionelle kovalente Immobilisierung von Signalmolekülen eignen. Dabei konnten keine starken Unterschiede bei der Reaktion der Zellen auf die Oberflächen PEMA oder POMA festgestellt werden. Auch die Einführung eines zusätzlichen Spacers war für die Signaltransduktion nach Stimulation mit kovalent immobilisiertem LIF nicht notwendig. Für künftige Arbeiten zur kovalenten Immobilisierung von LIF an MA-Copolymeren ist deshalb aus Stabilitäts- und Effizienzgründen die Oberfläche POMA zu bevorzugen. Diese Favorisierung kann jedoch aufgrund der unterschiedlichen Tertiärstruktur anderer Proteine und ihrer verschiedenen Steifigkeiten sowie bei der Verwendung anderer Zelltypen nicht automatisch für ein anderes Modellsystem übernommen werden. Die Verwendung hydrophiler Oberflächen oder die Kopplung über Spacer sollte demnach in Abhängigkeit vom zu immobilisierenden Protein und den auszusiedelnden Zellen geprüft werden. Die vorgestellte Kopplungsmethode umgeht die Modifikation des Proteins sowie Behandlungen zur Vernetzung des Zytokins. Die Immobilisierungsreaktion ist bei Raumtemperatur und Umgebungsdruck sowie unter sterilen Bedingungen durchführbar. Immobilisierte Zytokine werden homogen kovalent an der Oberfläche gebunden und sind dort für die Zellen zugänglich. Außerdem ermöglicht die Einstellung definierter Belegungsdichten die gezielte Applikation von Zytokindosen. MA-Copolymere sind somit nicht nur für die Kultivierung von Stammzellen unter Erhaltung des Stammzellstatus einsetzbar, sondern eignen sich auch für Differenzierungsstudien. Teilergebnisse dieser Arbeit wurden publiziert unter K. Alberti, R.E. Davey, K. Onishi, S. George, K. Salchert, F.P. Seib, M. Bornhäuser, T. Pompe, A. Nagy, C.Werner, and P.W. Zandstra. Functional immobilization of signaling proteins enables control of stem cell fate. Nat Methods, 5(7):645–650, Jul 2008 und T. Pompe, K. Salchert, K. Alberti, P.W. Zandstra, and C. Werner. Immobilization of growth factors on solid supports for the modulation of stem cell fate. Nat Protocols, 5(6):1042–1050, Jun 2010
In vitro cultivation of (embryonic) stem cells requires a defined environment. Together different properties as cytokine supplement, extracellular matrix composition or topographic design can mimic this stem cell niche in an artificial system. For mouse embryonic stem cells the cytokine leukemia inhibitory factor (LIF) is able to keep those cells in undifferentiated state and to enhance self renewal without the supplement of other factors. In vivo LIF exists in both diffusible and extracellular matrix immobilized form. This work investigates whether LIF can be immobilized covalently to alternating maleic anhydride (MA)-copolymers in a functional manner. When bioavailable, covalently immobilized LIF should be able to interact with specific cytokine receptor subunits and provide information to keep murine embryonic stem cells in a pluripotent state. In aqueous solution with neutral pH (such as phosphate buffered saline, PBS) and ambient temperature and pressure MA-copolymers react spontaneously with aminogroups and therefore represent a useful support for covalent protein immobilization. Depending on the choice of co-monomer, properties of copolymer vary: ethylene results in hydrophilic poly-(ethylene-alt-maleic anhydride) (PEMA), octadecene in more hydrophobic poly-(octadecene-alt-maleic anhydride) (POMA). LIF can be covalently immobilized onto the MA-copolymers as shown by radiolabeling experiments. The amount of cytokine coupled to PEMA increased linear whereas on POMA saturation could be observed for higher concentrations. A subsequent coupling of a polyethylene glycol spacer (PEG7) further modified the properties and led to more hydrophilic surfaces. The amount of LIF per area decreased in comparison to MA-copolymers without the spacer but the graph characteristics remained unaltered (linear for PEMA+PEG7, saturation for POMA+PEG7). During the first three days in buffer solution supplemented with bovine serum albumin, unbound LIF was displaced and the amount of immobilized cytokine remained stable. This Stability after preincubation allowed to immobilize required amounts of LIF per area. Although hydrophilic surfaces with PEMA showed swelling behavior resulting in increased layer thickness after incubation in PBS, accessibility to LIF for an antibody was not impaired. The amounts per area detected by radiolabeling method and using the antibody were similar and indicated that LIF was not covered by copolymers. For cell culture addition of diffusible as well as immobilized growth factors or cytokines requires dosage control. Frequently it is necessary to provide homogeneous distribution of the factor of interest. In the present study analysis by fluorescence microscopy confirmed homogeneity for surfaces with covalently immobilized LIF (iLIF) but not for LIF physisorbed to extracellular matrix components collagen type I and fibronectin. LIF transduces signals via the JAK/STAT pathway. Preliminary experiments with LIF-sensitive fibroblasts showed similar activation of STAT3 after stimulation with immobilized or diffusible LIF. The results of STAT3 activation revealed an activation profile with high intensities within the first 15 minutes for both immobilized and diffusible LIF followed by decrease. STAT3 activation profiles were similar on different surfaces and independent of LIF presentation mode. These results revealed that fibroblasts could recognize covalently immobilized LIF onto MA-copolymers and were able to activate STAT3. In the absence of LIF mESC start to differentiate within 24 to 36 hours and loose their pluripotency. To confirm the functional immobilization of LIF mouse embryonic stem cells (mESC) were cultivated on iLIF-modified POMA or POMA+PEG7 surfaces for 72 hours and stained for activated STAT3. Results showed a dose-dependent activation increasing with the iLIF amount per area. Higher amounts (8 and 75 ng/cm^2) of iLIF activated STAT3 similar to 10 ng/ml diffusible LIF. Introduction of PEG7 spacer did not further increased STAT3 activation. Both, the amount of ESC marker Oct4 and the percentage of Oct4-positive cells increased with higher amounts of iLIF and showed similar results as obtained with 10 ng/ml diffusible LIF. Murine ESC cultivated on LIF physisorbed to matrix components expressed similar amounts of transcription factor Oct4 compared to unstimulated cells. STAT3 activation and Oct4 expression in the absence of diffusible cytokine indicated a functional covalent immobilization of LIF. To confirm the pluripotency, mESC were stimulated for 6 to 8 subcultures only with iLIF, cell aggregates were fused with mouse embryos and implanted in pseudopregnant surrogate mothers. Three weeks after birth the contribution of mESC aggregates to chimera was evaluated. ESC stimulated with iLIF only contributed to chimera formation with around the same frequency as mESC cultivated with 10 ng/ml diffusible LIF. Thus, iLIF maintained pluripotency of mESC during in vitro expansion and could replace diffusible LIF. As shown by the experiments, MA-copolymers provide a support to covalently immobilize cell signaling molecules in a functional manner. This method of coupling does not need any protein modification or cross-linking treatment after protein incubation. Reaction can be carried out under sterile conditions at ambient temperature and pressure. The immobilized ligand is distributed equally on the supporting copolymer and the adjustment of required ligand amounts is possible. These properties characterize MA-copolymers as a suitable support to immobilize cell signaling molecules not only for keeping the stem cell fate but also for differentiation studies. Parts of this work were published: K. Alberti, R.E. Davey, K. Onishi, S. George, K. Salchert, F.P. Seib, M. Bornhäuser, T. Pompe, A. Nagy, C.Werner, and P.W. Zandstra. Functional immobilization of signaling proteins enables control of stem cell fate. Nat Methods, 5(7):645–650, Jul 2008. T. Pompe, K. Salchert, K. Alberti, P.W. Zandstra, and C. Werner. Immobilization of growth factors on solid supports for the modulation of stem cell fate. Nat Protocols, 5(6):1042–1050, Jun 2010

About 30 % of all infertile couples suffer from infertility of an unexplained cause. Leukemia inhibitory factor (LIF) is a glycoprotein produced by the endometrium and is an important cytokine in the implantation process. LIF exerts its biological functions through heterodimerization of its two receptors: LIF receptor (LIFR) and gp130. Point mutations in the LIF gene have been associated with female infertility. The aim of this study was to investigate whether single nucleotide polymorphisms (SNPs) in the genes of LIFR and gp130 could cause reduced fertility in women. To this end, 115 samples from women diagnosed with unexplained infertility and 191 samples from fertile women were studied. Three SNPs in the gp130 gene and two SNPs in the LIFR gene were analyzed using real-time PCR. One significant difference and a tendency to difference were detected in the gp130 gene for women with unexplained infertility. There were no differences in the LIFR gene variations. In conclusion, polymorphisms in gp130, and thereby disturbances in the LIF pathway, could be one cause for infertility in women diagnosed with unexplained infertility.

Initial studies in this work explored the role of interleukin-6 (IL6) and leukemia inhibitory factor (LIF) in preimplantation bovine embryos. Neither cytokine affected the total percentage of embryos which developed to the blastocyst stage in vitro. However, supplementation of IL6 increased blastocyst inner cell mass (ICM) cell number without affecting trophectoderm (TE) cell number. Additionally, we found that IL6 activated signal transducer and activator of transcription 3 (STAT3) specifically within ICM cells. LIF, however, did not affect ICM cell number or activate STAT3 in ICM cells, and was not pursued further. This increase in ICM cell number by IL6 was largely comprised of hypoblast (GATA6+:NANOG-) cells, and most IL6-responsive cells in day 9 blastocysts were hypoblast cells (as measured by STAT3 activation). However, some epiblast (NANOG+) cells were also IL6-responsive, and IL6 appeared to initially slow epiblast differentiation. Finally, IL6-treated blastocysts also had increased transcripts of hypoblast/primitive endoderm (PE) markers. These results indicate that IL6 may improve pregnancy retention of IVP embryos by improving yolk sac development, but further work is needed to confirm this theory. Activation of STAT3 by IL6 could be blocked with a chemical Janus kinase 2 (JAK2) inhibitor (AZD1480). JAK2 inhibition from day 5 to 8 resulted in blastocyst ICMs with fewer than 10% the normal cell number, regardless of IL6 supplementation. This indicates that STAT3 is critical for bovine ICM development. Further analysis revealed that inhibition of JAK2/STAT did not prevent ICM formation but disrupted its maintenance. Additionally, we assessed the suitability of zinc sulfate and a bovine embryonic stem cell culture media (TeSR) for improving bovine embryo development in vitro. Zinc sulfate increased day 8 blastocyst total and ICM cell number. Therefore, zinc sulfate appears to improve blastocyst quality. The TeSR medium improved embryo development beyond day 8. In normal synthetic oviduct fluid, blastocysts degenerated after day 8, while blastocysts moved to TeSR had greatly increased cell numbers, and even exhibited PE migration out from the ICM, a phenomenon that has not been reported in vitro. This indicates that extended blastocyst culture is possible with TeSR media.
Doctor of Philosophy
Bovine embryos have been produced in vitro for the purpose of being transferred to recipient cattle to produce a calf since the 1980s. This practice allows cattle breeders to increase the number of offspring from their best females each year, and also allows for more rapid progress in generational genetic improvement. However, only approximately 10% of bovine oocytes survive and produce a calf. This poor efficiency of bovine in vitro embryo production negatively impacts the procedure's widespread use. A significant portion of these embryo losses are likely a result of inadequate in vitro culture conditions, particularly of the embryo culture media, the fluid in which embryos are grown. This media is often called "synthetic oviduct fluid," or SOF, because it is designed to mimic the fluid present in the cow's oviduct, where the embryo would normally reside. However, SOF is much simpler in nature than actual cow oviduct fluid, and this leads to reduced embryonic survival of in vitro produced embryos. Unfortunately, we know very little of what molecules control and promote bovine embryo development. Therefore, one major goal of bovine embryo research is to identify these factors and add them to SOF. The goal of this work was to examine the ability of three molecules, interleukin-6 (IL6), leukemia inhibitory factor (LIF), and zinc sulfate, to increase the number and quality of blastocysts produced through in vitro culture techniques. Additionally, I tested the replacement of SOF with a complex cell culture media, known as TeSR. This medium is more complex than SOF, and therefore should better promote embryo development. This work revealed that IL6, but not LIF, improves in vitro produced (IVP) bovine blastocyst quality. Unfortunately, neither IL6 nor LIF affected the percentage of embryos which survived to the blastocyst stage. However, IL6, but not LIF, increased the number of cells in the inner cell mass (ICM) of the blastocysts. ICM cells are the portion of the embryo which will produce the future calf. IVP bovine embryos are known to have fewer cells than normal, in vivo derived, blastocysts, and this issue is believed to cause some embryonic death after embryo transfer. Therefore, treatment with IL6 may increase the percentage of embryos which will survive after transfer and produce a calf. We also found the addition of zinc sulfate to SOF to benefit embryo quality. None of the concentrations of zinc significantly improved the percentage of embryos which survived to the blastocyst stage, but 2 µM zinc did increase ICM cell number. Like IL6, this may improve embryo survival after transfer. The use of the TeSR media as a replacement for SOF had some benefits. Unfortunately, this media is unusable for producing embryos for transfer to recipients, as we discovered early embryos could not survive in the media. However, blastocyst-stage embryos thrived in it, and could be cultured in vitro for a longer period of time as a result. Therefore, this media will be a useful tool for studying bovine embryo development in vitro, however it is unlikely to benefit calf production. In summary, this work provides evidence that zinc sulfate and IL6 are beneficial additions to SOF. However, future work is needed to determine if embryos produced with these factors are more able to produce a calf. Additionally, we discovered that TeSR is a superior extended blastocyst culture medium.

The mammalian testis is divided into two distinct compartments which carry out its principal functions. Spermatogenesis occurs within the seminiferous tubules and androgen biosynthesis primarily occurs in the interstitial space. Both these processes are entirely dependent upon the two major testicular somatic cell populations - the Sertoli and Leydig cells respectively. In human males, testicular spermatogenic and endocrine function declines during the ageing process. Of particular significance is the reported age-related decrease in Leydig cell androgen production as androgens have been suggested to play a crucial role in supporting lifelong general health in men, with low circulating testosterone linked to an increased risk of developing chronic age-related cardiometabolic diseases. However, the relationship between ageing, testicular function and disease is not fully understood, impeding the development of novel therapeutic strategies to treat age-related testicular dysfunction. In one set of studies undertaken herein, a series of novel mouse models of premature ageing were utilised to begin to dissect the process of age-related testicular degeneration. Firstly, a novel knockout-first conditional allele of a previously reported premature-ageing model driven by Cisd2 (CDGSH Iron Sulphur Domain 2) deficiency was validated and the testicular phenotype characterised and compared to that of naturally aged mice at 18-months of age. Histological analyses revealed premature testicular atrophy at 6-months of age in CISD2 deficient mice, consistent with observations of the naturally aged testis. Circulating testosterone was significantly lower in CISD2-deficient mice compared to wild-type controls at 6-months of age and the luteinising hormone/testosterone ratio was significantly elevated, indicative of compensated Leydig cell failure. mRNA expression of key genes involved in androgen production were also significantly reduced in the CISD2-deficient testis, pointing to Leydig cell dysfunction in this model of premature aging. Next, Cre/LoxP technology was used to delete Cisd2 from specific testicular cell populations to determine which cell types control/support Leydig cell function during the ageing process. Testosterone production was unaffected when Cisd2 was disrupted in either the Leydig cell population or Sertoli cell population. These observations suggest that disruption to the testicular microenvironment in which Leydig cells reside, rather than intrinsic Leydig cell ageing, may play a significant role in age-associated Leydig cell dysfunction. A second set of studies were carried out to investigate the role of leukemia inhibitory factor (LIF) signalling in the maintenance of testicular function. LIF is a pleiotropic cytokine belonging to the interleukin-6 family. In the rodent testis, LIF is expressed in fetal life and adulthood; the peritubular myoid cells thought to be the main site of production. Given their anatomical location within the testis, LIF produced by peritubular myoid cells may act on both intratubular and interstitial cells to influence spermatogenesis and steroidogenesis respectively. Indeed, LIFR is expressed in germ cells, Sertoli cells, Leydig cells as well as testicular macrophages suggesting that LIF may be a key paracrine regulator of testicular function. However, the precise role of LIF/LIFR signalling in the testis is largely unknown. As such, models of testicular cell-specific Lifr deletion were generated using Cre/LoxP technology. Analysis of these novel models of conditional LIFR ablation revealed that LIFR is dispensable in germ cells for normal spermatogenesis. However, LIFR ablation from Sertoli cells resulted in a progressive degenerative phenotype, characterised by abnormal germ cell loss, sperm stasis, seminiferous tubule distention and subsequent atrophy of the seminiferous tubules. In a final set of studies, a rat model of Leydig cell ablation-regeneration was used to determine the regenerative capacity of human adipose-derived perivascular stem cells (hAd-PSC) as a potential therapy for testicular dysfunction. Following ethane dimethanesulphonate (EDS) mediated Leydig cell ablation, primary hAd-PSCs, cultured with or without LH, IGF-1, PDGFBB, T3 and ITS supplement, were transplanted into the rat testis and Leydig cell regeneration was monitored via serial measurements of circulating luteinising hormone (LH) and testosterone. Overall, hAd- PSCs had no impact on the recovery of circulating testosterone levels. However, when pre-cultured with the cocktail of hormone/growth factor supplements, the LH spike induced by the removal of testosterone negative feedback was dampened, suggesting the transplanted cells may promote Leydig cell regeneration. Whether these cells differentiate into Leydig cells, or simply provide paracrine support to the regenerating Leydig cells remains to be determined. Although Ad-PSCs may enhance regeneration kinetics, the transplanted cells were undetectable in the testis 5 weeks post transplantation suggesting they may not survive in the context of long term xenogeneic transplantation.

Le stroma inflammatoire joue un rôle primordial lors de la carcinogénèse. Dans ce contexte, nous montrons que la cytokine LIF est à l'origine d'une population de fibroblastes capable de remodeler la matrice extracellulaire de manière à la rendre permissive à l'invasion collective des cellules tumorales. En effet, nous montrons que la production de LIF par les cellules tumorales et fibroblastiques, après une stimulation au TGFβ, va réguler les capacités contractiles et pro-invasives de ces dernières via la régulation du cytosquelette d'acto-myosine et de manière indépendante de l'expression de α-SMA. En effet, l'inhibition pharmacologique des kinases JAKs permet de bloquer l'environnement fibrotique des tumeurs et d'ainsi bloquer l'invasion des cellules tumorales in vitro et in vivo. Nous montrons ensuite que LIF est à l'origine d'un switch épigénétique responsable de l'activation constitutive de la voie de signalisation JAK1/STAT3. Ce processus, régulé par la forme acétylée de STAT3, et son interaction avec l'ADN methyltransférase DNMT3b permet l'hypermethylation du promoter de la phosphatase SHP1 et donc la phosphorylation constitutive de JAK1. Une fois mis en place, ce nouveau profil de méthylation est maintenu par DNMT1. La surexpression de LIF dans les carcinomes humains corréle avec un environnement fibrotique, la présence de nodules invasifs et un mauvais pronostic clinique. De même, il existe une forte corrélation négative entre l'acétylation de STAT3 et l'expression de SHP1 dans le stroma tumoral. Nos résultats montrent qu'inhiber l'activité des DNMT et des kinases JAK permet de reprogrammer les capacités pro-invasive des fibroblastes associés aux carcinomes
Signaling crosstalk between tumor cells and fibroblasts confers proinvasive properties to the tumor microenvironment. We identify LIF as a tumor promoter that mediates proinvasive activation of stromal fibroblasts independent of alpha-smooth muscle actin expression. We demonstrate that a pulse of transforming growth factor β (TGF-β) establishes stable proinvasive fibroblast activation by inducing LIF production in both fibroblasts and tumor cells. In fibroblasts, LIF mediates TGF-β-dependent actomyosin contractility and extracellular matrix remodeling, which results in collective carcinoma cell invasion. Indeed, pharmacological inhibition of JAK activity by counteracts fibroblast-dependent carcinoma cell invasion in vitro and in vivo. We next unveil that LIF initiates an epigenetic switch leading to the constitutive activation of JAK1/STAT3 signaling, which results in sustained pro-invasive activity of fibroblasts. The process is mediated by p300-histone acetyltransferase acetylation of STAT3, and DNA methyltransferase DNMT3b, which induce the hypermethylation of SHP1 phosphatase promoter and results in constitutive phosphorylation of JAK1. Sustained JAK1/STAT3 signaling is maintained by DNMT1. Accordingly, carcinomas display strong LIF upregulation, which correlates with dense collagen fiber organization, cancer cell collective invasion, and poor clinical outcome. Moreover, we show that STAT3 acetylation and phosphorylation are inversely correlated with SHP1 expression in tumors stroma. Combined inhibition of DNMT activities and JAK signaling results in long-term reversion of CAF-associated pro-invasive activity and restoration of the wild-type fibroblast phenotype

Le stroma inflammatoire joue un rôle primordial lors de la carcinogénèse. Dans ce contexte, nous montrons que la cytokine LIF est à l'origine d'une population de fibroblastes capable de remodeler la matrice extracellulaire de manière à la rendre permissive à l'invasion collective des cellules tumorales. En effet, nous montrons que la production de LIF par les cellules tumorales et fibroblastiques, après une stimulation au TGFβ, va réguler les capacités contractiles et pro-invasives de ces dernières via la régulation du cytosquelette d'acto-myosine et de manière indépendante de l'expression de α-SMA. En effet, l'inhibition pharmacologique des kinases JAKs permet de bloquer l'environnement fibrotique des tumeurs et d'ainsi bloquer l'invasion des cellules tumorales in vitro et in vivo. Nous montrons ensuite que LIF est à l'origine d'un switch épigénétique responsable de l'activation constitutive de la voie de signalisation JAK1/STAT3. Ce processus, régulé par la forme acétylée de STAT3, et son interaction avec l'ADN methyltransférase DNMT3b permet l'hypermethylation du promoter de la phosphatase SHP1 et donc la phosphorylation constitutive de JAK1. Une fois mis en place, ce nouveau profil de méthylation est maintenu par DNMT1. La surexpression de LIF dans les carcinomes humains corréle avec un environnement fibrotique, la présence de nodules invasifs et un mauvais pronostic clinique. De même, il existe une forte corrélation négative entre l'acétylation de STAT3 et l'expression de SHP1 dans le stroma tumoral. Nos résultats montrent qu'inhiber l'activité des DNMT et des kinases JAK permet de reprogrammer les capacités pro-invasive des fibroblastes associés aux carcinomes
Signaling crosstalk between tumor cells and fibroblasts confers proinvasive properties to the tumor microenvironment. We identify LIF as a tumor promoter that mediates proinvasive activation of stromal fibroblasts independent of alpha-smooth muscle actin expression. We demonstrate that a pulse of transforming growth factor β (TGF-β) establishes stable proinvasive fibroblast activation by inducing LIF production in both fibroblasts and tumor cells. In fibroblasts, LIF mediates TGF-β-dependent actomyosin contractility and extracellular matrix remodeling, which results in collective carcinoma cell invasion. Indeed, pharmacological inhibition of JAK activity by counteracts fibroblast-dependent carcinoma cell invasion in vitro and in vivo. We next unveil that LIF initiates an epigenetic switch leading to the constitutive activation of JAK1/STAT3 signaling, which results in sustained pro-invasive activity of fibroblasts. The process is mediated by p300-histone acetyltransferase acetylation of STAT3, and DNA methyltransferase DNMT3b, which induce the hypermethylation of SHP1 phosphatase promoter and results in constitutive phosphorylation of JAK1. Sustained JAK1/STAT3 signaling is maintained by DNMT1. Accordingly, carcinomas display strong LIF upregulation, which correlates with dense collagen fiber organization, cancer cell collective invasion, and poor clinical outcome. Moreover, we show that STAT3 acetylation and phosphorylation are inversely correlated with SHP1 expression in tumors stroma. Combined inhibition of DNMT activities and JAK signaling results in long-term reversion of CAF-associated pro-invasive activity and restoration of the wild-type fibroblast phenotype

Self-renewal of mouse embryonic stem (mES) cells is dependent upon the presence of leukemia inhibitory factor (LIF). LIF induces tyrosine phosphorylation and nuclear translocation of STAT3 (signal transducer and activator of transcription 3) which is thought to promote self-renewal by inducing key target genes. The molecular chaperone heat shock protein 90 (Hsp90) is involved in signal transduction pathways and regulates STAT3 activity in different cell types. However, the role of Hsp90 in regulating STAT3 activity in mES cells has not previously been investigated. The aim of this study was to investigate if Hsp90 interacts with STAT3 in mES cells and to determine if this interaction is important for the maintenance of self-renewal. It was found that when mES cells were cultured for 24.0 hours in the absence of LIF, the expression levels of total STAT3, tyrosine-phosphorylated STAT3 (pYSTAT3), and the pluripotency marker, Nanog, were down regulated. However, the expression level of Hsp90 was found to be slightly up-regulated over the same period. Significantly, it was found that the amount of STAT3 in differentiating mES cells available for binding to Hsp90 was decreased upon down-regulation of STAT3 by LIF withdrawal. Therefore, STAT3-Hsp90 interactions in mES cells were dependent on the presence of LIF, which suggested that the reduction in STAT3-Hsp90 interaction may have resulted from the low levels of STAT3. Despite a dramatic reduction in the expression levels of pYSTAT3 upon 24.0 hours of culture of mES cells in the presence of the STAT3 tyrosine phosphorylation inhibitor, cucurbitanin I, there was no obvious reduction in the levels of total STAT3, Oct-3/4 or Nanog. These results suggested that the levels of unphosphorylated STAT3 rather than pYSTAT3, maybe more important in the maintenance of mES cells self-renewal.

STAT5A et B sont des facteurs de transcription qui constituent le point de convergence de nombreux signaux extracellulaires. Parmi leurs fonctions biologiques, ils sont connus pour leur rôle dans le développement et la différentiation des cellules hématopoïétiques. Cependant, un taux d’activation et/ou d’expression élevé de ces protéines aboutit à une prolifération incontrôlée des cellules aboutissant ainsi à une leucémogenèse. Ce présent travail vise à caractériser des aptamères ADN (Apta1 et Apta2) sélectionnés préalablement au sein de notre laboratoire contre STAT5B afin de réguler son activité dans le contexte leucémique. Les aptamères ADN sont des oligonucléotides simple brin qui adoptent une structure 3D et interagissent de manière spécifique avec leurs cibles. Contrairement aux anticorps, ils sont peu immunogènes ; ils possèdent alors un potentiel thérapeutique intéressant. La première partie de ce projet se focalise sur l’étude de la capacité d’Apta1 et Apta2 à interagir avec la forme cellulaire et recombinante de STAT5B par pull down et calorimétrie à titrage isotherme. La seconde partie concerne l’évaluation de l’activité d’Apta2 par l’étude de son effet sur la viabilité d’un modèle de leucémie myéloïde chronique et sur sa capacité à perturber la voie de signalisation impliquant STAT5
STAT5A and B are common transcription factors that constitute a convergent point for many cellular pathways. Among their multiple biological functions, they are well known in promoting immune cell development and differentiation. When some oncogenic mutations occur, STAT5A and B are highly activated leading to uncontrolled proliferation and then to leukemia. Thus, they constitute a prime target to therapeutic intervention. In this work, we characterize new DNA aptamers (Apta1 and Apta2) selected previously by our laboratory against STAT5B. DNA aptamers are single stranded DNA molecules that can adopt 3D structures and recognize specific targets. Unlike antibodies, they fail to induce the immune response: they emerge as potentiel therapeutic molecules. In the first part of this work, the selected aptamers were assessed on their ability to interact with the cellular and recombinant form of STAT5B by using pull down assay and Isothermal Titration Calorimetry. In the second part, we focused on evaluating the effect of Apta2 on chronic myeloid leukemia cell line. For this purpose, cell viability, apoptosis process and JAK-STAT5 signaling pathway were depicted when cells are treated with Apta2

ABSTRACT

A defect implantation process is the major reason for unexplained infertility. Estrogen andprogesterone are steroid hormones preparing the endometrium for implantation. They mediatetheir effect through their receptors: estrogen receptor alpha and beta and progesteronereceptor A and B, respectively. Leukemia inhibitory factor (LIF), which is also important forimplantation, mediates its effect through LIF receptor and the coreceptor, gp130, and is downregulated by suppressors of cytokine signaling 1. The aim of the study was to compare thelevels of the steroid hormone receptors and LIF related factors in the endometrium of twogroups of women with the diagnosis unexplained infertility: one that became pregnant afterassisted reproduction and one that did not become pregnant. Before treatment of thesewomen, endometrial mRNA was collected during the window of implantation in themenstrual cycle. The levels of specific mRNAs were measured with real-time PCR. Womenwho had become pregnant had a significantly higher level of steroid hormone receptors. Thus,these proteins seem to be important for a pregnancy and may be suitable as receptivitymarkers.

Oligodendrocytes (OL)s are the dominant cell type in the white matter and are integral for synaptic transmission essential for proper neuronal communication between brain areas. Previous studies have shown that intravenous administration of the mononuclear fraction of human umbilical cord blood (HUCB) cells in rat models of stroke reduced white matter injury, gray matter injury and behavioral deficits. Yet the mechanisms used by HUCB cells remain unknown in ischemic injury. These studies will investigate both in vitro and in vivo approaches to elucidate this mechanism in OLs. When mature primary OLs were coincubated with HUCB cells, HUCB cells secreted soluble factors that reduced cell death in OLs exposed to OGD. Microarray analysis revealed that HUCB cell treatment induced OL gene changes. These changes included genes involved in cell proliferation, signaling, anti-oxidant activity, and myelination. To extend these findings, the middle cerebral artery occlusion (MCAO) model was used to assess the expression profile of protein products of gene changes observed in vitro. The in vivo data mirrored in vitro data in that metallothionein 3 (Mt3), peroxiredoxin 4 (Prdx4), myelin oligodendrocyte glycoprotein (Mog), U2AF homology kinase 1(Uhmk1), and insulin induce gene 1(Insig1) were upregulated in OLs of the white matter tract adjacent to the infarct. Furthermore, double immunofluorescence staining determined that OLs expressed these proteins. Other reports have shown that HUCB cells secrete soluble factors related to cellular protection, including interleukin 6 (IL-6), interleukin 8 (IL-8), and interleukin 10 (IL-10). Other factors are known for their proliferative actions, such as vascular endothelial growth factor (VEGF), BDNF, platelet derived growth factor B (PDGF-B), leukemia inhibitory factor (LIF), and granulocyte colony stimulating factor (GCSF) all of which converge on the Akt survival pathway. Given these findings we hypothesize that Akt activation is integral to HUCB cell mediated OL protection. In models of excitotoxicity, the addition of Akt inhibitor IV blocked HUCB cell mediated protection in OL cultures exposed to 24 hrs OGD. In vivo, HUCB cell treatment increased Akt activation, antioxidant protein expression and decreased caspase 3 cleavage in the external capsule in a time dependent manner. The next series of experiments determine whether the soluble factors secreted by HUCB cells could replace HUCB cells as treatment. LIF expression is increased in HUCB cells as compared to peripheral blood and as previously mentioned, LIF is secreted by HUCB cells. Additionally, LIF rescued OLs from spinal cord and experimental autoimmune encephalomyelitis injury. Thus LIF was investigated. LIF protected OL subjected to 24 hr OGD, increased antioxidant Prdx4 gene expression and reduced reactive oxygen species production. Additionally the inclusion of Akt inhibitor IV blocked LIF induced OL protection. Similar results were obtained when GCSF was evaluated. All these findings indicate that HUCB cell mediated OL/white matter protection is due to the soluble factors secreted by the mononuclear population of these cells. These soluble factors including LIF activate cellular machinery leading to enhanced cellular survival. Here we found a specific survival pathway activated by soluble factors released from HUCB cells, leading to Akt activation. Akt activation arrests stroke induced apoptosis and reduced the expansion of the infarct, promoting functional recovery from acute ischemic injury.

Orientadores: Liliana Aparecida Lucci de Angelo Andrade, Fatima Aparecida Böttcher-Luiz
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: A invasão do endométrio pelo trofoblasto extraviloso é fundamental no desenvolvimento do feto e da placenta, processo este controlado por fatores ligados à atividade imunológica e hormonal que, quando alterada, pode resultar em interrupção da gestação e/ou geração das chamadas doenças trofoblásticas gestacionais. Em algumas situações, pode haver evolução para o coriocarcinoma, neoplasia maligna do trofoblasto, em que há evidências da atuação das moléculas ligadas ao processo de fusão celular e inflamação. Porém, os estudos neste tema são incipientes e inconclusivos. Considerando essas informações, o objetivo deste trabalho é estudar de forma comparativa a expressão das citocinas LIF, IL-6 e do ativador de transcrição STAT-3, além da telomerase, em material de aborto, de placenta normal a termo e de coriocarcinoma. Métodos: a expressão destas moléculas foi avaliada pelos métodos: imunoistoquímica (IHQ), imunofluorescência (IF), Western Blotting (WB) e Real-Time PCR (RT-PCR), em amostras de material de aborto, placenta normal a termo e coriocarcinoma (N=12 cada um). Os ensaios de WB e Real-Time PCR empregaram material a fresco de placenta normal a termo e seu cultivo celular e cultura da linhagem BeWo. Resultados: no material de aborto, as reações de IHQs evidenciaram expressão moderada de IL-6 em 58,4% dos casos e intensa de STAT-3 em 33,3%. Na placenta normal, observou-se intensa marcação de IL-6 em 50% e de STAT- 3 em 16,7% dos casos, enquanto que, no coriocarcinoma, houve expressão intensa de IL-6 em 50% e de STAT-3 em 75% dos casos. Por outro lado, as reações para LIF tiveram expressão nula em todos os três grupos. Pelo WB houve expressão proteica de IL-6 apenas no material fresco de placenta normal e ausência de expressão na sua cultura primária e na linhagem BeWo; LIF não foi expresso em todos os grupos estudados. STAT-3 foi detectado no citoplasma em todos os grupos, entretanto, a expressão nuclear da STAT-3 fosforilada (pSTAT-3) não foi observada na IF e nem pelo WB. Na análise gênica pelo RTPCR houve forte expressão de IL-6 e STAT-3 no material fresco de placenta normal e expressão muito fraca na cultura primária de placenta normal e na linhagem BeWo; a expressão de LIF foi muito fraca em todos os grupos. Apenas a linhagem BeWo demonstrou forte expressão gênica da telomerase, contrastando com a completa falta de expressão no material fresco de placenta normal e em sua cultura primária. Conclusão: A intensa expressão IHQ de IL-6 e STAT-3 no coriocarcinoma indica a atuação de ambas na carcinogênese. A expressão proteica de IL-6 no material fresco de placenta normal e sua ausência no material de cultura primária e na linhagem BeWo pode ser ocasionado pelo contato célula-a-célula nas culturas aderentes, inibindo o crescimento celular e, consequentemente, as vias de sinalização. A falta de expressão da pSTAT-3 tanto na IF como por WB demonstra que a via JAK-STAT está sendo desativada. A ausência de expressão de LIF, em todos os métodos estudados, sugere que esta citocina poderia estar sendo inibida por meio de proteínas SOCS3 ou, atuando, de modo indireto, na proliferação celular do coriocarcinoma. O aumento da atividade da telomerase nas células BeWo reforça sua relação com o fenótipo maligno e a aponta como um bom marcador para progressão da doença
Abstract: The invasion of the endometrium by extravillous trophoblast is a fundamental process in the growth of the fetus and placenta. The process is controlled by factors related to the immune and hormonal activity that, when changed, may result in termination of pregnancy and development of so-called gestational trophoblastic diseases. In some cases, changes can result in malignancy, in which some molecules play a role in cell fusion process and inflammation, although studies in this area are inconclusive. Considering this information, the study had the aim of investigating the expression of cytokines LIF, IL-6, STAT- 3 and the function of telomerase to understand their participation in abortion, in normal at term placenta and choriocarcinoma. Methods: The expression of the molecules was assessed by immunohistochemical assay (IHC), immunofluorescence (IF), Western Blotting (WB) and Real-Time PCR (RT - PCR) using fixed material from biopsies of abortions, normal at term placentas and choriocarcinoma along with fresh tissue of normal at term placenta and their primary culture and BeWo cell line. Paraffin embedded material used in IHC and IF assays were obtained from the Department of Pathology files. Tests of WB and Real-Time PCR employed fresh material, obtained from cell cultures of normal at term placenta and the BeWo line. Results: IHC reactions to abortion biopsies showed moderate staining for IL-6 in 58.4% of cases and intense for STAT-3 in 33.3 % of cases. In biopsies of normal placenta, there was intense reaction for IL-6 in 50% of cases, intense for STAT-3 in 16.7%; choriocarcinoma showed intense staining for IL- 6 in 50% of cases and also for STAT-3 in 75% of cases. On the other hand, LIF expression was missing in all three groups. WB analyses showed IL-6 protein in fresh material from normal placentas, but no expression in placenta primary cultures and BeWo line. LIF was absent in all groups. Cytoplasmic STAT-3 was observed in all groups, while the nuclear expression of phosphorylated STAT-3 was absent. On gene analyses a strong expression of IL-6 and STAT- 3 was observed from fresh normal placenta, but very weak expression in primary cultures of normal placenta and BeWo cell line. LIF expression was very weak in all groups. In regard to the gene expression of telomerase, it was strong in the BeWo line which contrasted with its complete lack of expression in fresh normal placenta and its primary culture. Conclusion: The high expression of IL-6 and STAT-3 in biopsies of choriocarcinoma indicates the role of both in tumor progression. Regarding protein expression, the presence of IL-6 in the material from fresh normal placenta, and its absence in primary culture and BeWo line may be caused by the cell-to-cell contact cultures by inhibiting cell growth and thus signaling pathways. However, the lack of expression of phosphorylated STAT-3 whether through IF or WB shows that its JAK-STAT pathway is inhibited. Lack of expression of the LIF suggests that it might be involved indirectly in choriocarcinoma cell proliferation or be inhibited by SOCS3 protein. Moreover, the increased telomerase activity of BeWo cells enhances their relation to the malignant phenotype and indicates a good marker for disease progression
Doutorado
Ciencias Biomedicas
Doutora em Ciências Médicas

Base teórica: O processo de implantação do embrião no ser humano é extremamente complexo e, ao mesmo tempo, essencial para que a mulher possa engravidar. Neste processo, em que o endométrio precisa sofrer uma série de mudanças para tornar-se receptivo, a adequada expressão de MFG-E8 (milk fat globule epidermal growth factor 8), seu receptor a integrina αvβ3 e LIF (leukemia inhibitory factor) parecem ter um papel importante. Além do mais, mulheres com infertilidade e endometriose podem apresentar a falha de implantação como uma grande barreira para obter seu sucesso terapêutico. Objetivos: Avaliar o papel de MFG-E8 e do seu receptor integrina αvβ3 em um modelo de implantação in vitro com uma linhagem celular trofoblástica e outra de epitélio endometrial. Comparar a expressão de MFG-E8, de integrina αvβ3 e de LIF no endométrio de pacientes férteis e inférteis com endometriose durante a janela de implantação. Métodos: No primeiro ensaio, utilizando-se uma linhagem celular bem diferenciada de adenocarcinoma de endométrio (células Ishikawa) e uma linhagem de coriocarcinoma de trofoblasto, o modelo in vitro de implantação humana foi estabelecido. Para investigação do impacto do bloqueio de MFG-E8 e integrina αvβ3, ambas linhagens celulares foram pré-tratadas com anticorpos contra estas proteínas em diferente concentrações antes do ensaio de adesão. No ensaio subsequente, para comparar a expressão de MFG-E8, de integrina αvβ3 e de LIF no endométrio humano, foram realizadas biópsias no período da janela de implantação (LH+7 a LH+10) com cateter de Pipelle. As amostras foram submetidas a imunohistoquímica, e analisadas através do HSCORE. Resultados: Na avaliação in vitro observamos que as células Ishikawa pré-tratadas com anticorpo anti-MFG-E8 causaram diminuição da adesão das esferas Jar dose-dependente. Por outro lado, o pré-tratamento das esferas Jar não resultou em diminuição significativa da adesão. Pré-tratamento com anticorpos anti-integrina αvβ3, tanto de células Ishikawa como de esferas Jar, causaram inibição significativa, dose-dependente, da adesão das esferas. A análise imunohistoquímica das biópsias realizadas durante a janela de implantação mostrou uma expressão aumentada de MFG-E8 em pacientes com endometriose e infertilidade. Além do mais, houve expressão diminuída de LIF no grupo em estudo. Contudo, não houve diferença estatisticamente significativa na expressão de integrina αvβ3 entre os grupos em estudo. Conclusão: Este estudo demonstrou que, quando se bloqueia MFG-E8 ou seu receptor integrina αvβ3 em células Ishikawa em um modelo in vitro, ocorre uma diminuição de adesão das células Jar. Além do mais, bloqueando-se a integrina αvβ3 nas esferas Jar, também ocorre uma diminuição da adesão destas nas células Ishikawa. No entanto, quando estudamos o endométrio in vivo de pacientes com endometriose e infertilidade, encontramos a expressão aumentada de MFG-E8 e diminuída de LIF durante a janela de implantação no endométrio.
Background: The human implantation process is very complex and, at the same time, it is essential for women to achieve pregnancy. In this process, where the human endometrium must go through a lot of changes in order to become receptive, an adequate expression of MFG-E8 (milk fat globule epidermal growth factor 8), integrin αvβ3 and LIF (leukemia inhibitory factor) appear to play an important role. Furthermore, women with endometriosis and infertility may have in their implantation process the key to achieve pregnancy. Objectives: To investigate the role of MFG-E8 and its receptor integrin αvβ3 in the attachment of trophoblast cells to the endometrial epithelium, in an in vitro model. To compare endometrial expression of MFG-E8, integrin αvβ3 and LIF between fertile patients and patients with endometriosis and infertility during the window of implantation. Methods: In our first assay, by using a well-differentiated endometrial adenocarcinoma cell line (Ishikawa cells) and choriocarcinoma human trophoblast cells (Jar cells), an in vitro model mimicking human implantation was established. To investigate the impact of blocking MFG-E8 and integrin αvβ3, the cell lines were pretreated with antibodies against those proteins at different concentrations before the attachment assay. Moreover, to compare endometrial expression of MFG-E8, integrin αvβ3 and LIF, endometrial biopsies were performed during the window of implantation (LH+7 to LH+10) with the Pipelle catheter. The samples were submitted immunochemistry, and analyzed with HSCORE. Results: Pretreatment of Ishikawa cells with anti-MFG-E8 antibody caused a dosedependent and significant inhibition of attachment is our in vitro assay. On the other hand, pretreatment of Jar spheroids did not result in a significant effect on the attachment rate. Pretreatment of Ishikawa cells as well as Jar spheroids with anti-integrin avb3 antibodies resulted in a dose-dependent, significant inhibition of attachment. The immunochemistry analysis of the endometrial biopsies performed during the window of implantation showed increased MFG-E8 expression in patients with endometriosis and infertility. Moreover, there was lower LIF expression in the study group. Conclusion: This study showed that blocking MFG-E8 and its receptor integrin αvβ3 in Ishikawa cells diminishes Jar spheroid attachment in an in vitro model. Moreover, blocking integrin αvβ3 in the trophoblastic cells also diminished their attachment to the Ishikawa monolayer. Nevertheless, when we studied the endometrium of patients with endometriosis and infertility, we saw an increased expression of MFG-E8 and decreased expression of LIF during the window of implantation.

Axonal degeneration is a major pathological feature of the central nervous system (CNS) inflammatory demyelinating disease multiple sclerosis (MS). This axonal degeneration has major consequences, as functional axonal regeneration in the CNS is largely absent. Cumulative axonal degeneration is the likely cause of the majority of progressive MS-related disability, and therefore, the need for novel neuroprotective therapies for MS exists. Experimental autoimmune encephalomyelitis (EAE), an animal model of MS pathology, also produces axonal injury. In particular, the optic nerve and spinal cord are key sites of neuroinflammation in mouse EAE. By utilizing this model, the short term and long term effects of the putative neuroprotective cytokine, leukaemia inhibitory factor (LIF), were investigated in the optic nerve and spinal cord utilising a number of outcome measures of axonal dysfunction. These included MRI measures of water diffusivity along (ADC ||) and across (ADC┴) the optic nerves, serum levels of phosphorylated neurofilament heavy chain subunit (pNF-H) and histological morphometric measures. LIF treatment reduced EAE grade and pNF-H plasma levels, decreased ADC┴, but had no effect on ADC ||, axon counts or inflammatory infiltration.
In contrast, genetic deletion of LIF and its sister cytokine ciliary neurotrophic factor (CNTF), not only increased EAE grade and pNF-H levels, but also decreased optic nerve ADC|| and optic nerve and spinal cord axon densities. After reviewing current literature, we hypothesize that the target cell for endogenously upregulated LIF in EAE may be the neuron or axon, whereas the target cell for exogenously administered therapeutic LIF may be another cell type, possibly infiltrating macrophages and activated microglial cells. LIF antagonist treatment did not have any affect on EAE grade, pNF-H levels or MRI parameters. This lack of effect may be due to the inability of the LIF antagonist to enter the CNS, supporting the hypothesis that endogenous LIF has a centrally acting mechanism.

Le LIF est un facteur protéique dont l'action biologique est pléiotropique in vitro et in vivo. En effet, en fonction du type de cellules cibles, cette action varie de la survie et/ou la prolifération à la différenciation. En particulier, le LIF induit la survie et la prolifération des cellules souches embryonnaires de souris (les cellules es). La technologie de recombinaison homologue dans ces cellules nous a permis d'obtenir une lignée de souris mutantes pour le LIF, appelées souris LIF-/-, dans laquelle le gène LIF n'est plus fonctionnel. Plusieurs fonctions biologiques différentes sont altérées dans cette lignée. En effet, le taux de cellules souches hématopoïétiques y est significativement réduit et le fonctionnement des cellules t, de neurones sympathiques et l'implantation à la surface de l'utérus maternel y sont défectueux. Nous pensons que le LIF participe au maintien en survie des cellules souches hématopoïétiques in vivo. Par ailleurs, le LIF agit sur ses cellules cibles par le truchement d'un récepteur composé du LIFR et de la gp 130. La gp 130 transmet le signal LIF à l'intérieur de la cellule. Afin d'élucider la nature du signal intracellulaire active par la gp 130 dans les cellules souches, nous inactivons le gène gp 130 dans les cellules es, et ceci de façon totale et conditionnelle par recombinaison homologue.

Evidências epidemiológicas associam diferentes fatores ambientais, tais como poluição e ingestão de alimentos contaminados, com desfechos gestacionais negativos e fertilidade diminuída em humanos. Não há duvidas de que a poluição do ar nos grandes centros urbanos é capaz de provocar desfechos negativos sobre a gestação: baixo peso ao nascer, prematuridade, perda gestacional, entre outros. Entretanto, poucos estudos foram conduzidos para avaliar um possível efeito da exposição à poluição ambiental particulada do ar sobre a saúde reprodutiva feminina. O objetivo deste trabalho foi avaliar se a exposição subcrônica a poluição atmosférica particulada da cidade de São Paulo é capaz de alterar a receptividade uterina à implantação embrionária. Para tanto, foram avaliados 3 grupos de fêmeas de camundongos (n=10), expostas cronicamente desde o período de desmame (PND21) até atingirem a idade reprodutiva (PND60) à duas concentrações de MP2,5 (600?g/m3 ou 1200ug/m3) ou ar filtrado. Diferentes parâmetros relacionados à fertilidade e a receptividade uterina foram avaliados. Nossos achados mostram que a exposição ao material particulado de origem veicular provoca alterações na ciclicidade estral prévia ao acasalamento, bem como um aumento no peso dos ovários. Avaliação da reserva folicular indica que há um aumento na quantidade de folículos médios associado à exposição a menor concentração de MP (p=0,04). A avaliação histopatológica do tecido uterino revelou que há aumentos na fração de volume das glândulas uterinas (600ug/m3; p=0,01); o epitélio glandular (p=0,001) e luminal (p=0,03) estão espessados e o diâmetro médio das glândulas uterinas foi maior nos grupos expostos ao MP (p=0,004). A análise qualitativa da distribuição de pinopódios no epitélio luminal por microscopia eletrônica de varredura e transmissão indica que há uma redução na presença destas estruturas. A avaliação da expressão de LIF por imunomarcação mostrou-se reduzida no epitélio luminal (p<0,001), nas glândulas (p<0,001) e estroma (p=0,004) nas fêmeas expostas ao MP, porém nenhuma diferença foi observada na expressão de MUC-1 (mucina). Entretanto quando avaliadas a expressão gênica de MUC-1 e LIF no tecido uterino e os níveis de IL-1beta e IL-6 no fluído uterino nenhuma diferença foi observada entre os grupos. Com base em nossos achados conclui-se que a exposição à poluição particulada do ar de origem veicular pode estar envolvida no aumento das perdas gestacionais e/ou implantacionais pelo comprometimento da receptividade uterina provavelmente pelo prejuízo do remodelamento uterino necessário a implantação
Epidemiological evidences have shown that environmental factors, such as environmental pollution and ingestion of contaminated food, are associated with negative gestational outcomes and decreased fertility in human. There is no doubt that exposure to air pollution in large urban areas are capable of impairing health (e.g. hypertension) and of aggravating preexisting diseases (e.g asthma). However, the effects of air pollution exposures on female reproductive health are lesser known. Previous experimental studies have shown that low birth weights are reduced and embryonic implantational index are reduced in animals exposed to ambient levels of air pollution. The aim of this study was to evaluate if sub chronic exposures to particulate air pollution before pregnancy and during the initial stages is capable to alter the uterine receptivity of mice. To test this, 3 groups of female mice were continually exposed from 21st to 60th postnatal day to either filtered or two different doses of concentrated ambient particles (MP2,5 - 600ug/m3 or 1200ug/m3) with the aid of a Ambient Particle Concentrator and different parameters associated with fertility and uterine receptivity were evaluated. Or data have shown that exposures to particulate air pollution from vehicular origin are associated to changes in estrous ciclicity, cycles are shorter and the number of days in estrous reduced. Evaluation of the follicular reserve also indicates that animals exposed to MP present an increased number of ovarian medium follicles (p=0.04). Histopathological evaluation of the uterine tissue revealed increases in the volume fraction of uterine glands (p= 0.01) of those animals exposed to 600ug/m3. The luminal (p= 0.03) and glandular epithelium (p= 0,001) are thicker and the uterine glands diameters (p=0,004) were greater in exposed animals. Qualitative analysis by transmission and scanning electron microscopy indicates that there is a reduction in the presence of pinopódios in the luminal epithelium of PM exposed females. The expressions of LIF assessed by immunohistochemistry in those females exposed to PM were reduced in the luminal epithelium (p<0,001), and in the glandular (p<0,001) and stromal compartments as well. However no differences in the expression of MUC-1 were seen. Gene expression of LIF and MUC-1 in the whole endometrium (qPCR) and the expression of IL-6 and IL-1beta in the uterine fluid did not show significant difference between the groups tested. In conclusion, our data have shown that exposures to ambient air particulate pollution can be associated with increased rates of implantational losses due to changes in the uterine receptivity related to factors involved in uterine remodeling for pregnancy

Les cancers, qu’il s’agisse de leucémies ou de tumeurs solides, sont le résultat de proliférations cellulaires anormales et non contrôlées au sein des tissus. Ces proliférations anarchiques sont le reflet d’une surexpression et/ou sur-activation de protéines intracellulaires engendrées par un événement oncogénique. Aujourd’hui encore il est donc nécessaire de trouver de nouvelles molécules à usage thérapeutique ciblant spécifiquement ces protéines. C’est dans ce contexte que les facteurs de transcription STAT5 constituent de véritables cibles de choix puisque ces protéines participent activement à la leucogénèse. L’implication directe des protéines STAT5 dans la génèse des leucémies a été démontrée par l’utilisation de formes mutées constitutivement active de STAT5. Les facteurs de transcription STAT5 jouent un rôle essentiel dans la voie de signalisation JAK/STAT. Cette voie aboutit à la régulation de grandes fonctions biologiques telles que la prolifération cellulaire, la différenciation cellulaire ou encore l’apoptose. L’objectif de ce projet consiste donc à cibler spécifiquement les protéines STAT5 dans le but de rétablir le processus de mort cellulaire et empêcher la prolifération des cellules cancéreuses. Les inhibiteurs spécifiques des protéines STAT5 sont sélectionnés selon la méthode SELEX qui permet d’isoler des ligands structurés de forte affinité pour la protéine. L’affinité et la spécificité de ces inhibiteurs, appelés aptamères, sont caractérisées à partir de modèles cellulaires de leucémies dépendant de l’activité des facteurs de transcription STAT5. Les aptamères sont aujourd’hui de véritables outils thérapeutiques en pleine évolution
Leukemias are due to abnormal cell proliferation, which is the result of intracellular over-expression or excessive activation of protein due to oncogenic event. Still today, it is necessary to find new therapeutic molecules, which specifically target these proteins. STAT5, via the JAK/STAT signaling pathway, controls fundamental cellular processes, including .cell survival, proliferation and differentiation. To struggle against tumorigenesis, JAK/STAT signaling pathway has to be inhibited. The aim of this project is to target specifically STAT5 factors to restore healthy signal transduction. We generated aptamers by an iterative in vitro selection. Aptamers are short-structured single strand DNAs or RNAs that bind with high affinity and specificity to their target. Once STAT5B recombinant proteins are produced, they are subjected to SELEX process. The number of rounds depends on various parameters. After seven rounds, two sequences are retrieved. The specificity and affinity of these aptamers are assessed by fluorescent immunoassays. Binding affinity and kinetics of interaction are characterized by SPR. Aptamer anti proliferative effects are determined by evaluation of the growth of cells depending on STAT5. Finally, we developed several .assays aiming at understanding the mechanism of an aptamer action on STAT5B such as phosphorylation measurement and EMSA. Aptamers are now emerging therapeutic tools; they exhibit significant advantages relative to protein therapeutics

Conselho Nacional de Desenvolvimento Científico e Tecnológico
The manipulation of oocytes enclosed in follicles Ovarian Pre-Antral, allows increasing the reproductive potential of females from the isolation and cultivation. The follicular viability can be affected negatively correlated with age, malnutrition, exposure to radio or chemotherapy and health conditions. The alternatives are to use these research for development of culture media which provides the maintenance of viability allows the activation and follicular development in vitro, for subsequent fertilization. Physiologically various growth factors are involved in the process of folliculogenesis, many of which have so far not been tested in goats. Among them the Sphingosine 1-phosphate (S1P) and Leukemia Inhibitory Factor (LIF), since, follicular development were obtained with these factors in other species, allowing follicular growth and ant apoptotic effect on ovarian tissue. The aim of this study was to evaluate the addition of different concentrations of S1P and the LIF to culture media separately, evaluating the maintenance of viability, activation and follicular development. Thus, the preantral follicle cultured with S1P in situ LIF for 7 days and follicular development as possible to maintain viability, activation and growth of preantral follicle in vitro. Concluding that the S1P 1 ng/ml LIF and 10ng/ml, were the concentration that the maintenance of viability and follicular activation of in situ and grown in vitro for 7 days improved the viability, activation and preantral follicular growth.
A Manipulação de Oócito Incluso em Folículos Ovariano Pré-Antral (MOIFOPA) possibilita o aumento do potencial reprodutivo das fêmeas a partir do isolamento e cultivo de FOPA. A viabilidade folicular pode sofrer influências negativas com a idade, desnutrição, exposição a rádio ou quimioterápicos e condições sanitárias adversas. As alternativas para aproveitamento desses FOPA são pesquisas voltadas para elaboração de meios de cultivos que proporcione o manutenção da viabilidade a possibilite a ativação e o desenvolvimento folicular in vitro, para posterior fertilização. Fisiologicamente vários fatores de crescimento estão envolvidos durante o processo da foliculogênese, muitos dos quais até o momento não foram testados em cabras. Dentre eles, a Esfingosina 1-fosfato (S1P) e o Fator Inibidor da Leucemia (LIF), uma vez que, o desenvolvimento folicular foram obtido com esses fatores em outras espécies, possibilitando crescimento folicular e efeito anti-apoptótico no tecido ovariano. O objetivo desse trabalho foi avaliar a adição de diferentes concentrações de S1P e o do LIF separadamente aos meios de cultivo, avaliando a manutenção da viabilidade, ativação e desenvolvimento folicular. Dessa forma, os FOPA cultivados in situ com S1P e LIF por 7 dias possibilitaram desenvolvimento folicular conforme a manutenção da viabilidade, ativação e crescimento dos FOPA in vitro. Concluindo que a S1P 1ng/ml e o LIF 10ng/ml, foram as concentração que condicionaram a manutenção da viabilidade e a ativação dos FOPA in situ cultivados in vitro por 7 em dias melhor mantiveram a viabilidade, ativação e crescimento folicular.

Em nosso trabalho anterior mostramos que dentre as citocinas analisadas, os níveis do mRNA de LIF nas células estromais pediátricas, de SMD e de SMD-LMA foram maiores quando comparados às células estromais de crianças saudáveis. No presente estudo, observamos um aumento tempo dependente nos níveis da proteína LIF após adição de SFB em todas as células analisadas (células estromais de crianças saudáveis, de SMD e de SMD-LMA). O envolvimento de p38, ERK e JNK na expressão LIF nestas células foi determinado pelo uso de inibidores dos membros das proteínas quinase ativadas por mitógenos: ERK (PD98059), p38 (SB302580) e JNK (SP600125) os quais inibiram a produção de LIF nas células estromais de crianças saudáveis, após estas serem estimuladas por SFB. No entanto, os níveis da expressão de LIF-induzido por soro nas células estromais de SMD e de SMD-LMA tratadas com SB302580 (p38) foram significativamente diminuídos, em comparação com a inibição observada no tratamento com PD98059 e SP600125 (p <0001, teste ANOVA). Em adição analisamos as formas fosforiladas de p38 e ERK, após 48hs na ausência ou na presença de soro por diferentes tempos. Níveis de atividade de ERK e do p38 foram inicialmente elevados na ausência de soro. A atividade de p38 foi sustentada após tratamento com SFB, entretanto, ERK apresentou uma variação de atividade durante o tratamento. Sugerimos que a sinalização das MAPKs (p38, ERK e JNK), em resposta a fatores de crescimento presentes no soro, parece desempenhar um papel importante na expressão da LIF em células estromais de crianças saudáveis, mas a sinalização do p38 parece ser funcionalmente mais importante nas mielodisplasias ou naquelas associadas à LMA
Our previous report showed that among the cytokines analysed, LIF mRNA levels in stromal cells from pediatric MDS and MDS-AML were higher as compared to those found in healthy stromal cells. In the present study, we have observed an increased protein LIF levels in a time dependent manner after FCS stimulation in all stromal cells analysed (MDS, MDS-AML and healthy children) and the involvement of p38, ERK and JNK pathways in the LIF expression in these cells was determined. In stromal cells from two healthy children, LIF production was equally inhibited in a dose dependent manner after FCS stimulation by mitogen-activated protein kinase (MAPKs) members inhibitors: ERK (PD98059), p38 (SB302580) and JNK (SP600125). However, in MDS and MDS-AML stromal cells, the levels of LIF-induced by serum, were significantly decreased by SB302580, as compared with the inhibition observed by treatment with PD98059 and SP600125 (p <0,001, ANOVA test). In addition we have analysed the presence of p38 and ERK phosphorylated forms in stromal cells, after 48hs of serum starvation or in the presence of FCS for different times. Activated ERK and p38MAPK levels were initially elevated in the absence of serum. p38MAPK activation was sustained after treatment with FCS, whereas ERK presented a variation of the activated forms during treatment. We suggest that the signalling of the MAPKs (p38, ERK and JNK) in response to growth factors present in the serum, seems to play an important role in the LIF expression by stromal cells of healthy children, but p38 MAPK signalling appears to be functionally more important in MDS and MDS-AML

碩士
中國醫藥學院
醫學研究所
87
Application of Recombinant Leukemia Inhibitory Factor (r-LIF) upon Reproduction INTRODUCTION: Leukemia inhibitory factor (LIF) is a pleiotropic cytokine of the interleukin-6 family and has different biological actions in various tissue systems. LIF can regulates the growth and differentiation of embryonic stem cells, primordial germ cells, peripheral neurons, osteoblasts, adipocytes, and endothelial cells. It is also crucial for successful implantation of the embryo in mice. The role of LIF in reproduction will be discussed. ABSTRACT Objective: To assess the effect of human recombinant leukemia inhibitory factor (LIF) in in the prolonged culture of fresh mouse embryos (Experiment 1) and human cryopreserved-thawing embryos (Experiment 2). The influence of different concentration of LIF in the in-vitro development of mouse embryos was also evaluated (Experiment 3). Materials and Methods: Experiment 1: Female CB6F1 mice that were between 6 and 8 weeks old were superovulated by 10 IU pregnant mare*s serum gonadotropin (PMSG) and 10 IU HCG intra-peritoneally; then mated with BDF1 male mice. Mice were divided randomly into three Groups, which included 1 Group of in vivo (Group 1) and 2 Groups of in vitro study (Group 2, 3). In Group 1 (control Group), mice were killed after HCG injection 116-120 hours. In Group 2 and 3, mice were killed after HCG injection 44-48 hours. The 2-cell embryos (Group 2, 3) and blastocyst stage embryos (Group 1) were washed by flush medium from the ampulla after the excision of the oviduct. In Group 2 (HTF + HSA), embryos were co-cultured with 30ml microdroplets of human tubal fluid (HTF) +0.5% human serum albumin (HSA). In Group 3 (HTF + HSA+ r-LIF): mouse embryos were co-cultured with HTF and r-LIF (1000 IU/ml) under paraffin oil. The embryonic numbers in different stage including 4-8 cell, morula, blastocyst, expanded blastocyst, and hatching blastocyst were recorded and compared. Experiment 2: After thawing, all human embryos were divided into four groups: (1) Human tubal fluid (HTF); (2). HTF + LIF (1000 IU/ml LIF); (3) M3TH medium; (4) M3TH medium + LIF (1000 IU/ml LIF). In the following 5 days post thawing, the embryo development in each groups were compared. Experiment 3: All 2-4 cell embryos of Female CB6F1 mice were culture in the human tubal fluid (HTF) media which contains different concentration of LIF. Mouse embryos were divided into 7 groups: (1) HTF; (2) 1500 IU/ml LIF; (3) 1000 IU/ml LIF; (4) 750 IU/ml LIF; (5) 500 IU/ml LIF; (6) 250 IU/ml LIF; (7) 125 IU/ml LIF. The embryonic numbers of different stages including 5-8 cell, 9-16 cell, morula, blastocyst and hatching blastocyst were recorded. Results: Experiment 1: Similar embryos development to 4-8 cell and morula stages were noted between Group 2 and 3 (87.3% vs. 91.0%; 74.6% vs. 87.1%, respectively). Further embryo development in blastocyst, expanded, and hatching blastocyst in Group 2 (48.1%, 31.7%, 18.5%) were lower than that of Group 3 (83.6%, 53.7%, 37.8%)(p<.05). Experiment 2: There were non-different in the early embryo development (2-4 cell to 9-16 cells) between each group. There was non-different between group 1 and 3 (2-4 cell to morula) and group 2 and 4 (2-4 cell to blastocyst). There was lower morula formation in group 1 than other groups and the lower blastocyst formation in group 1 and 3 when compare with group 2 and 4 were noted. Experiment 3: The percentage of early embryo stage (2-cell embryos to 6-16 cell stages) in each groups were non-significant different. There were higher formation rate of pre-implantation embryos (morula to hatching blastocyst) in group 2, 3, 4 and 5 than those in group 1, 6 and 7. Conclusion: R-LIF does not provide the obvious stimulation upon the early development of mouse and human embryos. R-LIF has positive effects on pre-implantation blastocyst growth, differentiation and hatching of mouse and human embryos. The r-LIF supplemented HTF could provided a similar culture environment for thawing embryos as M3 TH medium. The lowest concentration of r-LIF which could result in the optimal embryo development is 500 IU/ml. Keywords: r-LIF, leukemia inhibitory factor, coculture

Errata inserted.
Includes bibliographical references.
xi, 119, [75] leaves, [21] leaves of plates : ill. (chiefly col.) ; 30 cm.
This study demonstrates a conserved structural organisation of the mouse LIF gene that produces three differentially localised proteins. This provides a mechanism for specific control of the sites of LIF action and mechanisms for mediating the variety of putative actions for the LIF gene. Intracellular localisation of the LIF protein provides another example of intracellular cytokine action, mediated by a novel mechanism and provides a system for separate analysis of intracellular and extracellular cytokines.
Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry,1998?

碩士
中國醫藥學院
醫學研究所
89
Part 1. Recombinant Leukemia Inhibitory Factor (r-LIF) Enhance Pre-implantation Mouse Embryo Development In Vitro Objective: To assess the effect of human recombinant leukemia inhibitory factor (r-LIF) in mouse embryos. Materials and Methods: Female CB6F1 mice that were between 6 and 8 weeks old were superovulated by 10 IU pregnant mare‘s serum gonadotropin (PMSG) and 10 IU HCG intra-peritoneally; then mated with BDF1 male mice. Mice were divided randomly into three Groups, which included 1 Group of in vivo (Group 1) and 2 Groups of in vitro study (Group 2, 3). In Group 1 (control Group), mice were killed after HCG injection 116-120 hours. In Group 2 and 3, mice were killed after HCG injection 44-48 hours. The 2-cell embryos (Group 2, 3) and blastocyst stage embryos (Group 1) were washed by flush medium from the ampulla after the excision of the oviduct. In Group 2 (HTF + HSA), embryos were co-cultured with 30l microdroplets of human tubal fluid (HTF) +0.5% human serum albumin (HSA). In Group 3 (HTF + r-LIF): mouse embryos were co-cultured with HTF and r-LIF (1000 IU/ml) under paraffin oil. The embryonic numbers in different stage including 4-8 cell, morula, blastocyst, expanded blastocyst, and hatching blastocyst were recorded and compared. Results: Similar embryos development to 4-8 cell and morula stages were noted between Group 2 and 3 (87.3% vs. 91.0%; 74.6% vs. 87.1%, respectively). However, further embryo development in blastocyst, expanded, and hatching blastocyst in Group 2 (48.1%, 31.7%, 18.5%) were lower than that of Group 3 (83.6%, 53.7%, 37.8%). Conclusion : R-LIF does not provide the obvious stimulation upon the early development of mice embryo. However, r-LIF has positive effects on pre-implantation blastocyst growth, differentiation and hatching. Part 2. The effect of different concentrations of Recombinant leukemia inhibitory factor (LIF) on different development stage of mouse embryo in vitro Purpose: To assess the influence of different concentrations of recombinant human leukemia inhibitory factor (LIF) on the in-vitro development of mouse embryos. Materials and methods: The 2-4 cell embryos of CB6F1 mice were culture in the human tubal fluid (HTF) media containing different concentration of LIF. Mouse embryos were divided into 7 groups: (1) HTF; (2) 1500 IU/ml LIF; (3) 1000 IU/ml LIF; (4) 750 IU/ml LIF; (5) 500 IU/ml LIF; (6) 250 IU/ml LIF; (7) 125 IU/ml LIF. The embryonic numbers of different stages including 5-8 cell, 9-16 cell, morula, blastocyst and hatching blastocyst were recorded. Results: The percentage of early embryo stage (2-cell embryos to 6-16 cell stages) in all groups were non-significantly different. There were higher formation rates of pre-implantation embryos (morula to hatching blastocyst) in groups 2, 3, 4 and 5 than in groups 1, 6 and 7. Conclusion : LIF has positive effects on pre-implantation embryo development and has non-significant influence upon the early embryo development. The lowest concentration of LIF which could provide the optimal embryo development is 500 IU/ml. Part 3. Prolonged culture of human cryopreserved embryos with recombinant human leukemic inhibitory factor (rhLIF) Purpose: To evaluate the efficiency of recombinant human leukemic inhibitory factor (LIF) in the prolonged culture of human cryopreserved-thawing embryos. Patients and methods: After thawing, all embryos were divided into four groups: (1) Human tubal fluid (HTF); (2). HTF + LIF; (3) M3TH medium; (4) M3TH medium + LIF. In the following prolong culture, the embryo development in each groups were compared. Result(s): About the embryo development from 2-4 cell to 9-16 cell stage, there were non-different between each group. There was lower morula formation rate in group 1 (6.9%) than those in other groups (23.2%, 19.7%, 23.1%). The lower blastocyst formation in group 1 and 3 (0%, 0%) than those in group 2 and 4 (11.0%, 12.8%) were noted. Conclusion(s): LIF is beneficial for pre-implantation embryos. LIF does not influence the early embryo development. The LIF-supplemented HTF provided a similar culture environment for thawing embryos as M3 TH medium. Part 4. Leukemia Inhibitory Factor (LIF) expression in different endometrial location between fertile and infertile women throughout different menstrual phases Purpose: To demonstrate the leukemia inhibitory factor (LIF) expression in different endometrial locations between fertile and infertile women throughout different menstrual phases. The relationship between progesterone level and LIF expression were evaluated. Patients and methods: Idiopathic infertile and normal fertile women accepted the endometrial biopsies in follicular, periovulatory, and luteal phases. The luteal progesterone level was measured. Endometrial LIF immunostaining of luminal epithelium, glandular epithelium, and stroma were detected. The relationship between luteal LIF expression and progesterone level was evaluated. Results: Significant LIF expression was noted in the endometrium of fertile women than that of infertile women. The LIF expression was highest in the luminal epithelium, moderate in the glandular epithelium, and lowest in the stroma. The luminal and glandular epithelial staining were lowest in follicular phase, moderate in periovulatory phase, and strongest in luteal phase. The stromal LIF presented with a non-cyclical manner. The LIF expression is not related with the progesterone level. Conclusion: Endometrial LIF expression is related with human fertility. Endometrial LIF expression is dependent on cellular localizations and menstrual stages. Stronger LIF expression presents in the endometrial epithelium during luteal phase. Part 5. In-vivo gene transfer of leukemia inhibitory factor into mouse endometrium Objectives: Leukemia inhibitory factor (LIF) is important for embryogenesis and implantation. We aimed to transfect LIF gene into the mouse endometrium. Patients and methods: Expression plasmids carried LIF and luciferase genes for transfer. After superovulation, 100 ICR mice were mated with vasectomized mice. Then LIF-liposome (Group 1) and luciferase-liposome complexes (Group 2) were injected into their uterine lumen (day-0). Endometrial LIF and luciferase expressions were detected by reverse RT-PCR on day-0 to 4 post gene transfer. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control to normalize the gene transfection. Results: LIF mRNA and luciferase activities reached the peak expression on day-3. In Group 1, the ratios of LIF/GADPH on day-1 to 4 were 0.414, 1.096, 1.162, and 0.782. In Group 2, LIF/GADPH on day-1 to 4 were 0.24, 0.22, 0.35, and 0.32 Conclusions: Mouse endometrium could be effectively transfected with liposome-DNA mixtures. Endometrial LIF transfer via liposome may be effective in human.

碩士
中國醫藥學院
醫學類
84
Expression of Recombinant Human Leukemia Inhibitory Factor in Escherichia coliHuman leukemia inhibitory factor is a multi functional growth factor. The pET29b(+)expression vector includes S.Tag and His.Tag nucleotide sequence, encoding a 15-amino acid S-peptide and a histidine hexmar. Protein tagged with these two polypeptidesare easy to be detected and purified. For this reason, we utilize Escherichia coli toproduce recombinant human leukemia inhibitory factor in labatory. The results indicatethat rhLIF is expressed in a large amount in strain BLR(DE3)pLysS rather than in the other two tested strains. The production of rhLIF reaches to maximum at 3 hours after IPTG induction. The strategy to prepare rhLIF is briefly described below. The crudebacterica lysated was applied onto a His.Tag nickel-ion chelating column and the boundproteins were eluted with buffer containing 1M imidazole. The elute, however, is heterogenous. The S.Tag affinity column chromatography was therafter employed. The purity of target protein was chamatically improved albeit minor heterogeneity stillpersist. The proteins eluted from S.Tag column were then cleaved by thrombin and thenpass through a His.Tag column. Finally, the target protein can be purified to its homogenity. The yield is about 84.288 ug from 6 liters of BLR(DE3)pLysS culture. Thebiological activity of the purified protein was analysed as its proliferating activityon TF-1 cells. The LIF-like activity. However, was not detected. The possible reasons leading to lose activity of the purified proteins will be discussed in later part of this thesis.

博士
中山醫學大學
生物化學研究所
92
Good embryo development and receptive endometrium are essential factors for embryo implantation. Leukemia inhibitory factor (LIF) is an essential factor for implantation and establishment of pregnancy. However, its role in the development of preimplantation embryos remains controversial. The aim of this study is to assess the effects of LIF on development of preimplantation mouse embryo. We observe the relationship between the blastocyst morphology and the implantation rate for mice. Mouse blastocysts were then classified into 3 grades: grade I, small blastocysts; grade II, large blastocysts; grade III, hatching blastocysts. Although there was no significantly different in the implantation rates between the grade III and grade II, grade I was significantly decreased as compared with grade III. Grade I and grade II was also significantly decreased in both the diameter of blastocysts and cell number of inner cell mass (ICM) and trophectoderm (TE) as compared with grade III. These findings indicated that the expanded and haching blastocyst selections for embryo transfer in in vitro culture were evaluated with the high implantation rate. We successfully established the model of in vitro culture and blastocyst transfer for following experiments of LIF. Changes in preimplantation embryos were determined after microinjection of LIF antisense oligonucleotide at the two-pronucleus stage. The 0.5- or 1.0-fmol treated groups had significantly lower percentages of embryos developed to the morula or blastocyst stage and the 2.0-fmol treated group had significantly lower percentages of embryos developed to the four-cell, morula, or blastocyst stage. No embryos developed to the four-cell stage in the 4.0-fmol treated group. Moreover, there was a decreasing trend in the levels of LIF immunoactivity with the increasing amount of LIF antisense oligonucleotide injected. The diameter of blastocysts in the 2.0-fmol treated group was significantly smaller than that in the untreated group. The blastocysts in this group had significantly lower numbers of blastomeres and cells in the ICM or TE and ICM/TE ratio. The 1.0- or 2.0-fmol treated groups had significant lower implantation rates than their corresponding control groups. In the 2.0-fmol groups with supplementing exogenous LIF, significantly lower percentages were also observed in the four-cell, morula, and blastocyst stages. However, blastocysts treated with 50 ng/ml LIF had a significant higher percentage than those in the LIF gene impaired group without LIF supplement. These results indicate that LIF is a critical factor for the normal development of embryos at the preimplantation stages.

Für medizinische Anwendungen sind Stammzellen aufgrund ihrer Eigenschaften (Selbsterneuerung, hohe Proliferationsrate und Differenzierungsmöglichkeit in verschiedene Zelltypen) beispielsweise in den Bereichen des regenerativen Gewebeersatzes und der Zelltherapie sehr interessant. In vivo umgibt die Stammzellen eine definierte Mikroumgebung, die sie unterstützt sich zu teilen, ihren undifferenzierten Status aufrecht zu erhalten und Tochterzellen für das Wachstum, die routinemäßige Erneuerung oder den Ersatz von Gewebe zu produzieren. Diese Mikroumgebungen werden als Stammzellnischen bezeichnet. Für die Kultivierung von Stammzellen in vitro muss die in vivo-Situation möglichst getreu nachgestaltet werden. Ziel der Forschung ist die Schaffung einer künstlichen Umgebung, die sowohl die funktionellen Eigenschaften einer Nische besitzt als auch frei von Risiken xenogener Pathogene oder Gewebeunverträglichkeiten für die Anwendung am humanen Organismus eingesetzt werden kann. Einen Ansatz dafür bietet beispielsweise die Kopplung von Faktoren, die für den Erhalt der Stammzelleigenschaften notwendig sind, an synthetische Oberflächen. Ausgehend vom Bedarf an Kultur- oder Modellsystemen für die Expansion von (embryonalen) Stammzellen sollte in dieser Arbeit analysiert werden, ob alternierende Maleinsäureanhydrid (MA)-Copolymere ein geeignetes Trägersystem für die biofunktionelle kovalente Immobilisierung spezifischer Zytokine sind und dadurch unter anderem als künstliche Stammzellnische Anwendung finden können. MA-Copolymere eignen sich aufgrund ihrer spontanen Reaktion mit Aminogruppen für die Immobilisierung von Proteinen. Das Zytokin LIF (Leukemia inhibitory factor) existiert in vivo auch in immobilisierter Form und ist in embryonalen Mausstammzellen (mESC) allein in der Lage, das Stammzellpotential dieser Zellen zu erhalten. Aus diesem Grund ist LIF für die Analyse der Aufgabenstellung geeignet. Nach der Charakterisierung LIF-modifizierter Oberflächen wurde die biologische Verfügbarkeit des kovalent immobilisierten Zytokins mit Hilfe von LIF-sensitiven Fibroblasten und mESC der Linie R1 überprüft. Anschließend wurde im Mausmodell in vivo der Erhalt der Pluripotenz der mESC durch immobilisiertes LIF analysiert. Dafür standen die Oberflächen Poly(ethylen-alt-maleinsäureanhydrid) (PEMA) und Poly(octadecen-alt-maleinsäureanhydrid) (POMA) jeweils ohne und mit Polyethylenglykol (PEG7)-Modifizierung zur Verfügung, an die LIF kovalent gekoppelt wurde. Zusätzlich wurde LIF physisorptiv an einer Kollagen-Fibronektin-Matrix über hydrolysiertem POMA immobilisiert. Mit Hilfe von radioaktiv markiertem LIF konnte gezeigt werden, dass die Gesamtbeladungsmenge mit Zytokin von den Eigenschaften der MA-modifizierten Träger abhing. Auf PEMA konnten mit steigenden Immobilisierungskonzentrationen höhere Belegungsdichten an der Oberfläche erreicht werden, die im analysierten Bereich eine lineare Abhängigkeit zeigten. Aufgrund der starken Quellung in wässrigen Lösungen war eine Einlagerung von LIF-Molekülen in die Polymerschicht möglich und führte bei hohen Immobilisierungskonzentrationen auch nach 3 Tagen Inkubation mit proteinhaltigem Medium noch zur Verdrängung nicht kovalent gebundener Zytokinmoleküle aus PEMA-Oberflächen. Obwohl ein Teil des LIF in die Polymerschicht eindrang, war der Großteil der Moleküle für einen spezifischen Antikörper zugänglich. Hydrophobe Oberflächen mit POMA konnten bei hohen Immobilisierungskonzentrationen weniger LIF binden und zeigten Sättigungsverhalten der Oberflächen bei einer Belegungsdichte von 178 ng/cm^2 LIF. Eine Freisetzung von LIF nach mehr als 3 Tagen konnte nicht beobachtet werden. Gleichzeitig war hier aufgrund der hydrophoben Polymerseitenketten die Antikörperzugänglichkeit deutlich reduziert. Wegen des geringen Quellungsverhaltens von POMA in wässrigen Lösungen konnte eine Einlagerung des immobilisierten Zytokins in die Polymerschicht aber ausgeschlossen werden. Die kovalente LIF-Immobilisierung über PEG7-Spacer führte im Vergleich zu den nicht PEG-modifizierten Oberflächen PEMA und POMA zu jeweils geringeren Belegungsdichten, ohne dabei den Charakter der Abhängigkeit von der Immobilisierungskonzentration zu verändern (linear für PEMA+PEG7, Sättigung für POMA+PEG7). Die schlechte Antikörperzugänglichkeit von immobilisiertem LIF auf POMA konnte durch die Einführung des PEG7-Spacers deutlich verbessert werden und erreichte einen Wert ähnlich dem der hydrophilen PEMA-Oberflächen. Kovalent immobilisiertes LIF zeigte auf den vier MA-Oberflächen homogene und definiert einstellbare Belegungsdichten auf den einzelnen Proben. Die physisorptive Immobilisierung von LIF an extrazelluläre Matrixkomponenten auf hydrolysiertem POMA führte zu inhomogenen und bereits bei geringen Immobilisierungskonzentrationen instabilen Belegungsdichten. Die Einstellung definierter Belegungsdichten und die homogene Verfügbarkeit des Zytokins sind für die spätere Anwendung bei der Kultivierung wichtig, da so allen Zellen die gleiche definierte Zytokindosis unabhängig von der Oberflächencharakteristik präsentiert wird und Populationsunterschiede vermieden werden. LIF-sensitive Mausfibroblasten der Linie NIH3T3 reagierten auf immobilisiertes LIF mit der Aktivierung des Signalwegproteins STAT3. Durch den direkten Vergleich von STAT3-Aktivierungsprofilen nach Stimulation mit gelöstem oder immobilisiertem LIF konnte gezeigt werden, dass durch beide Präsentationsformen innerhalb der ersten 15 Minuten nach Stimulationsbeginn eine starke Aktivierung von STAT3 erfolgt, die anschließend wieder abklingt. Die Profile beider Präsentationsformen unterschieden sich in ihren Intensitäten nur bei der starken STAT3-Aktivierung. Dabei ergaben sich bei gelöstem LIF aufgrund der größeren Kontaktfläche mit Zytokin (gesamte Zelloberfläche) etwas stärkere Aktivierungen. Durch die sehr ähnlichen Aktivierungsprofile konnte nachgewiesen werden, dass das Zytokin LIF für Zellen zugänglich an MA-Copolymere mit und ohne Spacer-Modifizierung immobilisiert werden kann. Dabei lag ein Teil der Moleküle in einer Konformation und Orientierung gebunden vor, die die Funktionalität des Zytokins erhalten konnten. Zwischen den Oberflächen mit kovalenter LIF-Immobilisierung konnten keine wesentlichen Unterschiede in der STAT3-Aktivierung festgestellt werden. LIF war an all diesen Oberflächen für die LIF-sensitiven NIH3T3 Mausfibroblasten biologisch verfügbar. LIF-abhängige embryonale Mausstammzellen (mESC) reagierten nach 72 Stunden LIF-Stimulation mit der Aktivierung von STAT3. Bei Belegungsdichten ab 8 ng/cm^2 kovalent immobilisiertem LIF auf POMA mit und ohne PEG7-Spacer konnten ähnliche Aktivierungen wie durch die Stimulation mit gelöstem LIF festgestellt werden. Dies bestätigte die biofunktionelle LIF-Immobilisierung. Zwischen den POMA-Oberflächen mit und ohne PEG7 war dabei kein deutlicher Unterschied erkennbar. Eine reduzierte Zugänglichkeit des Antikörpers auf POMA beeinflusste demnach die biologische Verfügbarkeit des Zytokins für die mESC nicht. Der Erhalt des Stammzellpotentials durch kovalent an POMA gebundenes LIF konnte in vitro durch die Präsenz von Oct4 im Zellkern der mESC nachgewiesen werden. Durch die instabile Immobilisierung bei physisorptiver Assoziation des Zytokins an Matrixkomponenten über hydrolysiertem POMA reduzierte sich der Erhalt des Stammzellpotentials auf diesen Oberflächen stark. Kovalent immobilisiertes LIF dagegen konnte auch während der Kultur über mehrere Passagen hinweg die Pluripotenz der murinen ESC erhalten. Nach der Fusion mit Blastozysten beteiligten sich diese kultivierten Zellen in vivo erfolgreich an der Bildung von Chimären. Dabei konnten keine Unterschiede der Chimärenhäufigkeit zwischen der Kultivierung der mESC mit gelöstem oder kovalent an POMA immobilisiertem LIF festgestellt werden. Kovalent an MA-Copolymere immobilisiertes LIF ist demnach in der Lage, gelöstes LIF vollständig zu ersetzen und über mehrere Passagen hinweg allein das Stammzellpotential von mESC zu erhalten. Die Experimente zeigten, dass sich MA-Copolymere für die funktionelle kovalente Immobilisierung von Signalmolekülen eignen. Dabei konnten keine starken Unterschiede bei der Reaktion der Zellen auf die Oberflächen PEMA oder POMA festgestellt werden. Auch die Einführung eines zusätzlichen Spacers war für die Signaltransduktion nach Stimulation mit kovalent immobilisiertem LIF nicht notwendig. Für künftige Arbeiten zur kovalenten Immobilisierung von LIF an MA-Copolymeren ist deshalb aus Stabilitäts- und Effizienzgründen die Oberfläche POMA zu bevorzugen. Diese Favorisierung kann jedoch aufgrund der unterschiedlichen Tertiärstruktur anderer Proteine und ihrer verschiedenen Steifigkeiten sowie bei der Verwendung anderer Zelltypen nicht automatisch für ein anderes Modellsystem übernommen werden. Die Verwendung hydrophiler Oberflächen oder die Kopplung über Spacer sollte demnach in Abhängigkeit vom zu immobilisierenden Protein und den auszusiedelnden Zellen geprüft werden. Die vorgestellte Kopplungsmethode umgeht die Modifikation des Proteins sowie Behandlungen zur Vernetzung des Zytokins. Die Immobilisierungsreaktion ist bei Raumtemperatur und Umgebungsdruck sowie unter sterilen Bedingungen durchführbar. Immobilisierte Zytokine werden homogen kovalent an der Oberfläche gebunden und sind dort für die Zellen zugänglich. Außerdem ermöglicht die Einstellung definierter Belegungsdichten die gezielte Applikation von Zytokindosen. MA-Copolymere sind somit nicht nur für die Kultivierung von Stammzellen unter Erhaltung des Stammzellstatus einsetzbar, sondern eignen sich auch für Differenzierungsstudien. Teilergebnisse dieser Arbeit wurden publiziert unter K. Alberti, R.E. Davey, K. Onishi, S. George, K. Salchert, F.P. Seib, M. Bornhäuser, T. Pompe, A. Nagy, C.Werner, and P.W. Zandstra. Functional immobilization of signaling proteins enables control of stem cell fate. Nat Methods, 5(7):645–650, Jul 2008 und T. Pompe, K. Salchert, K. Alberti, P.W. Zandstra, and C. Werner. Immobilization of growth factors on solid supports for the modulation of stem cell fate. Nat Protocols, 5(6):1042–1050, Jun 2010.
In vitro cultivation of (embryonic) stem cells requires a defined environment. Together different properties as cytokine supplement, extracellular matrix composition or topographic design can mimic this stem cell niche in an artificial system. For mouse embryonic stem cells the cytokine leukemia inhibitory factor (LIF) is able to keep those cells in undifferentiated state and to enhance self renewal without the supplement of other factors. In vivo LIF exists in both diffusible and extracellular matrix immobilized form. This work investigates whether LIF can be immobilized covalently to alternating maleic anhydride (MA)-copolymers in a functional manner. When bioavailable, covalently immobilized LIF should be able to interact with specific cytokine receptor subunits and provide information to keep murine embryonic stem cells in a pluripotent state. In aqueous solution with neutral pH (such as phosphate buffered saline, PBS) and ambient temperature and pressure MA-copolymers react spontaneously with aminogroups and therefore represent a useful support for covalent protein immobilization. Depending on the choice of co-monomer, properties of copolymer vary: ethylene results in hydrophilic poly-(ethylene-alt-maleic anhydride) (PEMA), octadecene in more hydrophobic poly-(octadecene-alt-maleic anhydride) (POMA). LIF can be covalently immobilized onto the MA-copolymers as shown by radiolabeling experiments. The amount of cytokine coupled to PEMA increased linear whereas on POMA saturation could be observed for higher concentrations. A subsequent coupling of a polyethylene glycol spacer (PEG7) further modified the properties and led to more hydrophilic surfaces. The amount of LIF per area decreased in comparison to MA-copolymers without the spacer but the graph characteristics remained unaltered (linear for PEMA+PEG7, saturation for POMA+PEG7). During the first three days in buffer solution supplemented with bovine serum albumin, unbound LIF was displaced and the amount of immobilized cytokine remained stable. This Stability after preincubation allowed to immobilize required amounts of LIF per area. Although hydrophilic surfaces with PEMA showed swelling behavior resulting in increased layer thickness after incubation in PBS, accessibility to LIF for an antibody was not impaired. The amounts per area detected by radiolabeling method and using the antibody were similar and indicated that LIF was not covered by copolymers. For cell culture addition of diffusible as well as immobilized growth factors or cytokines requires dosage control. Frequently it is necessary to provide homogeneous distribution of the factor of interest. In the present study analysis by fluorescence microscopy confirmed homogeneity for surfaces with covalently immobilized LIF (iLIF) but not for LIF physisorbed to extracellular matrix components collagen type I and fibronectin. LIF transduces signals via the JAK/STAT pathway. Preliminary experiments with LIF-sensitive fibroblasts showed similar activation of STAT3 after stimulation with immobilized or diffusible LIF. The results of STAT3 activation revealed an activation profile with high intensities within the first 15 minutes for both immobilized and diffusible LIF followed by decrease. STAT3 activation profiles were similar on different surfaces and independent of LIF presentation mode. These results revealed that fibroblasts could recognize covalently immobilized LIF onto MA-copolymers and were able to activate STAT3. In the absence of LIF mESC start to differentiate within 24 to 36 hours and loose their pluripotency. To confirm the functional immobilization of LIF mouse embryonic stem cells (mESC) were cultivated on iLIF-modified POMA or POMA+PEG7 surfaces for 72 hours and stained for activated STAT3. Results showed a dose-dependent activation increasing with the iLIF amount per area. Higher amounts (8 and 75 ng/cm^2) of iLIF activated STAT3 similar to 10 ng/ml diffusible LIF. Introduction of PEG7 spacer did not further increased STAT3 activation. Both, the amount of ESC marker Oct4 and the percentage of Oct4-positive cells increased with higher amounts of iLIF and showed similar results as obtained with 10 ng/ml diffusible LIF. Murine ESC cultivated on LIF physisorbed to matrix components expressed similar amounts of transcription factor Oct4 compared to unstimulated cells. STAT3 activation and Oct4 expression in the absence of diffusible cytokine indicated a functional covalent immobilization of LIF. To confirm the pluripotency, mESC were stimulated for 6 to 8 subcultures only with iLIF, cell aggregates were fused with mouse embryos and implanted in pseudopregnant surrogate mothers. Three weeks after birth the contribution of mESC aggregates to chimera was evaluated. ESC stimulated with iLIF only contributed to chimera formation with around the same frequency as mESC cultivated with 10 ng/ml diffusible LIF. Thus, iLIF maintained pluripotency of mESC during in vitro expansion and could replace diffusible LIF. As shown by the experiments, MA-copolymers provide a support to covalently immobilize cell signaling molecules in a functional manner. This method of coupling does not need any protein modification or cross-linking treatment after protein incubation. Reaction can be carried out under sterile conditions at ambient temperature and pressure. The immobilized ligand is distributed equally on the supporting copolymer and the adjustment of required ligand amounts is possible. These properties characterize MA-copolymers as a suitable support to immobilize cell signaling molecules not only for keeping the stem cell fate but also for differentiation studies. Parts of this work were published: K. Alberti, R.E. Davey, K. Onishi, S. George, K. Salchert, F.P. Seib, M. Bornhäuser, T. Pompe, A. Nagy, C.Werner, and P.W. Zandstra. Functional immobilization of signaling proteins enables control of stem cell fate. Nat Methods, 5(7):645–650, Jul 2008. T. Pompe, K. Salchert, K. Alberti, P.W. Zandstra, and C. Werner. Immobilization of growth factors on solid supports for the modulation of stem cell fate. Nat Protocols, 5(6):1042–1050, Jun 2010.