Strategies to inhibit cell differentiation in long-term cultivation of embryonic stem cells D3 with regard to their use in the embryonic stem cell test

Deckblatt-Impressum Unterstützungshinweis Inhaltsverzeichnis Abbildungsverzeichnis Tabellenverzeichnis Einleitung Eigene Untersuchungen Ergebnisse Diskussion Zusammenfassung Summary Literaturverzeichnis Abkürzungsverzeichnis Anhang Danksagung Lebenslauf SelbständigkeitserklärungDer Embryonic Stem Cell Test (EST) ist ein in vitro Embryotoxizitätstest, mit dem Substanzen entsprechend ihrem embryotoxischen Potential klassifiziert werden können. Für die erfolgreiche Durchführung des EST ist die Verwendung undifferenzierter pluripotenter Stammzellen eine Grundvoraussetzung. Embryonalen Stammzellen (ES Zellen) wird eine nahezu unbegrenzte Vermehrungsfähigkeit zugesprochen, wenn sie bei Hemmung ihrer Differenzierung kultiviert werden. Allerdings kann es durch suboptimale Kulturbedingungen auch unter Differenzierungshemmung zu einer Differenzierung von ES Zellen und damit zu einer Veränderung der Zellpopulation kommen. In der vorliegenden Arbeit wurden verschiedene Methoden der Differenzierungshemmung für ES Zellen untersucht. Eine Methode wurde als geeignet bewertet, wenn sie den undifferenzierten Zustand der ES Zellen und deren Differenzierungskapazität zu Herzmuskelzellen erhalten kann. Dafür wurden die Stammzellen über einen Zeitraum von 50 Passagen unter dem Einfluss von 1000 U/ml bzw. 2000 U/ml Leukemia Inhibitory Factor (LIF), 20 ng/ml humanem Oncostatin M oder in Co- Kultur mit Feeder Layer aus STO-Fibroblasten mit und ohne Supplementierung des Kulturmediums mit 1000 U/ml LIF kultiviert. Über den Versuchszeitraum wurden die Zellen mit verschiedenen Methoden auf ihren Differenzierungsstatus untersucht. Neben der morphologischen Beurteilung der Zellen wurde auch die Expression der Stammzell-Marker SSEA-1 und Alkalische Phosphatase-Aktivität (AP-Aktivität) überprüft. Diese Marker werden typischerweise von undifferenzierten Stammzellen exprimiert und nach Differenzierung der Zellen herunterreguliert. Für den Nachweis wurden sowohl immunzytochemische, zytochemische und durchflusszytometrische Verfahren als auch ein photometrisch ausgewerteter Enzym-Aktivitätstest durchgeführt. Die Durchführung von an das Protokoll des EST angelehnten Proliferations- und Differenzierungstests ermöglichte die Untersuchung des Einflusses von Penicillin G und 5-Fluorouracil auf das Wachstums- und Differenzierungspotential der Stammzellen. Ziel der hier vorgelegten Arbeit war es zum einen, die verschiedenen Methoden der Differenzierungshemmung miteinander zu vergleichen und auf ihre Eignung für die Langzeitkultivierung von undifferenzierten ES Zellen zu überprüfen. Zum anderen sollten die verschiedenen Nachweismethoden für die SSEA-1-Expression bzw. die AP-Aktivität auf ihre Aussagekraft für die Beurteilung der Stammzellpopulation untersucht werden. Die Beurteilung erfolgte unter der Annahme, dass undifferenzierte embryonale Stammzellen in typischen Kolonien wachsen (bei Kultivierung in Zellkulturflaschen), das Oberflächenantigen SSEA-1 exprimieren und eine hohe Alkalische Phosphatase- Aktivität aufweisen. Im Differenzierungstest des EST können diese Zellen zu kontrahierenden Kardiomyozyten differenzieren. Es konnte gezeigt werden, dass bei Langzeitkultivierung die alleinige Supplementierung des Kulturmediums mit LIF eine Veränderung der Stammzellpopulationen mit zunehmendem Anteil differenzierter Zellen nicht verhindern konnte. Diese Zellpopulationen zeigten morphologische Veränderungen und wiesen einen verminderten Anteil SSEA-1-positiver Zellen, eine geringere AP-Aktivität und eine eingeschränkte Differenzierungskapazität zu Kardiomyozyten auf. Demgegenüber konnte durch direkte Co-Kultur der ES Zellen mit inaktivierten STO-Fibroblasten bzw. die Supplementierung des Kulturmediums mit Oncostatin M die Differenzierung der embryonalen Stammzellen über einen langen Zeitraum unterdrückt werden. Sowohl die Stammzell-Marker SSEA-1 und AP-Aktivität als auch die Differenzierungskapazität dieser Zellpopulationen blieben über den Untersuchungszeitraum auf einem hohen Niveau. Die bisherige Beschränkung der für den EST verwendeten ES Zellen auf einen Zeitraum von 25 Passagen ließe sich mit auf Feeder Layer kultivierten ES Zellen auf 50 Passagen erweitern und würde so eine bessere Ausnutzung von geeigneten Zellpopulationen ermöglichen. Für die Beurteilung der Zellpopulation im Hinblick auf ihre Eignung für die Durchführung des EST ist eine Quantifizierung der SSEA-1-positiven Zellen bzw. AP-Aktivität notwendig. Die Zell- bzw. Koloniemorphologie und die immunzytochemischen bzw. zytochemischen Nachweise von SSEA-1-Expression und Alkalischer Phosphatase-Aktivität der ES Zellen sind unsichere Methoden, den Differenzierungsstatus von embryonalen Stammzellen im Zellverband zu beurteilen. Auch die durchflusszytometrische Bestimmung der AP-positiven Zellen ermöglichte keine Vorhersage der Eignung einer Zellpopulation für die Durchführung des EST. Der durchflusszytometrisch bestimmte Anteil SSEA-1-positiver Zellen, ebenso wie die im Enzym-Aktivitätstest photometrisch bestimmte AP-Aktivität der Zellen, korrelierte hingegen mit der Differenzierungskapazität dieser Zellpopulationen. Die Untersuchungen der ES Zellen auf SSEA-1-Expression mittels Durchflusszytometrie oder auf Alkalische Phosphatase-Aktivität mit dem photometrisch ausgewerteten Enzym-Aktivitätstest bieten einfache und schnelle Methoden, über eine Quantifizierung dieser Stammzell-Marker eine Beurteilung der Stammzellpopulation vorzunehmen. Damit könnte die im Protokoll des EST bisher vorgeschriebene zeitaufwendige Qualitätskontrolle der ES Zellen ersetzt werden bzw. eine Überprüfung der Stammzellpopulation zeitnah zum EST erfolgen.The embryonic stem cell test (EST) is an in vitro embryotoxicity test that has been developed in order to classify test compounds for their teratogenic potential. The pluripotency of embryonic stem cells (ES cells) is a prerequisite for using these cells in the EST. If their differentiation is inhibited, the self-renewal-capacity of the embryonic stem cells is almost unlimited. However, suboptimal culture conditions can cause differentiation of stem cells even under differentiation inhibiting strategies and may result in a change of the cell population. This thesis is about the investigation of various methods of preventing ES cell differentiation. A method was looked upon positively if it allows to keep the undifferentiated status of the ES cells as well as their ability to differentiate into contracting cardiomyocytes. The permanent embryonic stem cell line D3 was cultivated over a period of 50 passages under the influence of 1000 U/ml Leukemia Inhibitory Factor (LIF), 2000 U/ml LIF, 20 ng/ml human Oncostatin M or in co-culture with STO-fibroblast-feeder layer with and without supplementation of the culture medium with LIF. Over the period of observation the cell populations were characterized for their differentiation status. In addition to the morphological investigation also the expression of the stem cell markers SSEA-1 (stage-specific embryonic antigen-1) and alkaline phosphatase activity (AP-activity) was investigated. These markers are typically expressed by undifferentiated stem cells and regulated down after the differentiation. Immunocytochemical and cytochemical techniques, FACScan-analyses and a photometrically determined enzyme-activity-test were performed. The influences of penicillin G and 5-fluorouracil on growth and differentiation of stem cells were investigated in proliferation and differentiation assays following the EST protocol. On the one hand, the presented study aimed on comparing the various differentiation inhibiting strategies and on checking their suitability for the long term cultivation of ES cells, on the other hand on the evaluation of different methods of determining the SSEA-1-expression or the AP-activity and their correlation with the ability of the ES cells to be used in the EST. The evaluation was based on the assumption that undifferentiated stem cells grow in typical colonies (when cultivated in culture flasks), that they express the surface antigen SSEA-1 and that they possess a high alkaline phosphatase activity. In the differentiation assay of the EST these cells are able to differentiate into contracting cardiomyocytes. The study demonstrated that in long term cultivation of ES cells the sole supplementation of the culture medium by LIF is not sufficient to inhibit changes of the stem cell population manifesting in increasing numbers of differentiated cells. These cell populations displayed morphological changes and a reduced number of SSEA-1 positive cells, a reduced AP- activity and a restricted capacity to differentiate into cardiomyocytes. In contrast the direct co-culture of ES cells with inactivated STO fibroblasts and the supplementation of the culture medium by Oncostatin M respectively, inhibited the differentiation for a long culture period. The stem cell markers SSEA-1 and AP-activity and the differentiation capacity of these cell populations remained on a high level. By using feeder layer the present restriction of the stem cell population to 25 passages in the EST could be extended to 50 passages. This would allow the better exploitation of a suitable cell population. A quantification of the SSEA-1 positive cells and the alkaline phosphatase activity respectively is required for the evaluation of the stem cell populations with regard to the performance of the EST. Of the various approaches the investigation of the colony morphology and immunocytochemical (SSEA-1 expression) or cytochemical analyses (AP-activity) proved to be no objective methods for evaluating the stem cell population according to their differentiation status. The suitability of the stem cell population for the EST is not predictable by the flowcytometric determination of AP-positive cells either. Whereas the flowcytometrically analyzed percentage of SSEA-1 positive cells as well as the photometrically determined AP-activity correlated with the differentiation capacity of the cell populations. FACScan- analyses for SSEA-1 expression or the enzyme activity test for AP-activity present a simple and fast way to evaluate the stem cell population by quantifying these stem cell markers. The time consuming quality check of the ES cells as prescribed in the EST protocol could be replaced and the test for suitability could be carried out shortly before the EST

Women in Alternatives

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