Nachweis von Nachkommen neuraler Stammzellen im Innenohr nach Blastozysteninjektion

Zusammenfassung

Hintergrund: Somatische Stammzellen besitzen ein großes Proliferations- und Differenzierungspotenzial. Eine Stammzelltherapie erscheint als Behandlungsansatz für Erkrankungen von Geweben mit bedingtem Regenerationspotenzial, wie dem Innenohr, vielversprechend. Gegenstand der vorliegenden Arbeit war die Untersuchung der Hörfunktion und Verteilung von neuralen Stammzellen im Innenohr nach Injektionen neuraler Stammzellen in Blastozysten.

Methoden: Neurale Stammzellen wurden aus den Kortexanlagen männlicher Mäuse am embryonalen Tag 14 isoliert und für vier Wochen in Neurobasalmedium kultiviert. Die so erhaltenen neuralen Stammzellen wurden in Blastozysten injiziert, die anschließend in Ammentiere transferiert wurden. Sechs Monate post partum erfolgten hirnstammaudiometrische Untersuchungen an sechs weiblichen Tieren. Zur Sichtbarmachung der Donorzellen wurde die Kochlea von zwei Tieren entnommen und LacZ gefärbt. Bei den vier übrigen Tieren erfolgte die Entnahme der Kochlea zur Gewebegewinnung, DNA-Extraktion und Amplifikation Y-chromosomal spezifischer DNA-Sequenzen zum Nachweis der injizierten Stammzellen im Innenohr.

Ergebnisse: Bei allen sechs Tieren ließen sich normale Hirnstammpotenziale ableiten. Bei drei von vier untersuchten Tieren war der Nachweis der Y-chromosomal spezifischen DNA-Sequenzen positiv.

Schlussfolgerung: Die Ergebnisse zeigen, dass Nachkommen neuraler Stammzellen nach Blastozysteninjektion im Innenohr nachweisbar sind und potenziell die Fähigkeit besitzen, sich in eine in der Entwicklung befindliche Kochlea zu integrieren ohne einen messbaren Hörverlust hervorzurufen.

Abstract

Background: Utilising the enormous proliferation and multi-lineage differentiation potentials of somatic stem cells represents a possible therapeutical strategy for diseases of non-regenerative tissues like the inner ear. In the current study, the possibility of murine neural stem cells to contribute to the developing inner ear following blastocyst injection was investigated. Methods: Fetal brain-derived neural stem cells from the embryonic day 14 cortex of male mice were isolated and expanded for four weeks in neurobasal media supplemented with bFGF and EGF. Neural stem cells of male animals were harvested, injected into blastocysts and the blastocysts were transferred into pseudo-pregnant foster animals. Each blastocyst was injected with 5 - 15 microspheres growing from single cell suspension from neurospheres dissociated the day before. The resulting mice were investigated six months post partum for the presence of donor cells. Brainstem evoked response audiometry (BERA) was performed in six animals. To visualize donor cells Lac-Z staining was performed on sliced cochleas of two animals. In addition, the cochleas of four female animals were isolated and genomic DNA of the entire cochlea was analyzed for donor contribution by Y-chromosome-specific PCR. Results: All animals had normal thresholds in brainstem evoked response audiometry. The male-specific PCR product indicating the presence of male donor cells were detected in the cochleas of three of the four female animals investigated. In two animals, male donor cells were detected unilateral, in one animal bilateral. Conclusion: The results suggest that descendants of neural stem cells are detectable in the inner ear after injection into blastocysts and possess the ability to integrate into the developing inner ear without obvious loss in hearing function.

Schlüsselwörter

somatische Stammzelle - Neurosphäre - Kochlea - Innenohrschwerhörigkeit - Stammzelltherapie

Key words

somatic stem cell - cochlea - neurosphere - sensorineural hearing loss - stem cell therapy

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