Untersuchung elternspezfischer Effekte beim Retinoblastom in einem in vitro Stammzellmodell
Zusammenfassung der Projektergebnisse
The childhood eye cancer retinoblastoma occurs after biallelic inactivation of the tumor suppressor gene RB1. As current models are insufficient for the analysis of early stages of tumor development and for the analysis of parent-of-origin effects in retinoblastoma, the goal was to generate an 3D organoid model for retinoblastoma using human pluripotent stem cells. For this hESCs with different mutations in the RB1 gene (in a heterozygous and homozygous state) were generated via the CRISPR/Cas9 system. Comparative differentiation, immunostainings and RNA sequencing were performed to analyze the potential of our retinoblastoma model which revealed promising results: The knock-out of RB1 in hESCs and the subsequent differentiation into neural retina results in the proliferation of cone photoreceptors which are the presumed cells-of-origin in retinoblastoma. Moreover, RB1ko retinal organoids show a gene expression pattern similar to the gene expression pattern in retinoblastoma tumors. Unfortunately, further differentiation beyond d152 is not possible due to the dissociation of the organoids. One possibility to overcome this issue could be optimizing the protocol as Rosanzka et al. do not observe dissociation of RB1ko retinal organoids with their protocol – at least this is not described in the article (Rozanska et al., 2022). Another approach could be to introduce the second mutation at a later time point during retina differentiation which might lead to more stable RB1ko retinal organoids and thus would make longer culturing possible. However, it might also be that pure organoid models are not suitable for the generation of a model system for retinoblastoma. Different studies showed the transplantation of cells of these organoids into eyes of mice resulting in retinoblastoma-like tumors thus possibly a combination of both would be better to model retinoblastoma. A further part of the project was the analysis of parent-of-origin effects in retinoblastoma using the established protocol. Different RB1 mutations exist resulting in differences in tumor development upon maternal or paternal inheritance which might be linked to genomic imprinting. To mimic the natural state of the RB1 locus in hESCs with fully methylated CpG85, CpG85 was exchanged by the EEF1A1 promoter on one allele. After the exchange expression of the alternative transcript RB1-E2B was observed. However, during culturing the exchanged EEF1A1 promoter showed a massive gain of methylation which results in silencing of the alternative RB1-E2B transcript. Thus, our approach failed as the CpG85 locus seems to be prone for methylation. We therefore tried to use iPSCs showing differential methylation at CpG85. As the differentiation into neural retina is a crucial step for this model, the differentiation potential of these iPSCs was tested before introducing the mutation resulting in parent-of-origin effects. However, the differentiation potential into neural retina using our protocol was highly insufficient in these cell lines. This is why we could not proceed with this part of the project. Cowan et al. showed that the differentiation potential of different iPSCs varies a lot. One could try to optimize the protocol for each iPS cell line. However, this is time consuming, expensive and maybe in the end again not successful. First, optimization is needed resulting in a unified and reliable model for retinoblastoma. Afterwards specific mutations causing retinoblastoma, and showing parent-of-origin effects, can be analyzed in more detail.
Projektbezogene Publikationen (Auswahl)
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Generation of heterozygous and homozygous hESC H9 sublines carrying inactivating mutations in RB1. Stem Cell Research, 33, 41-45.
Schipper, Leonie; Kanber, Deniz & Steenpass, Laura
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Biallelic and monoallelic deletion of the RB1 promoter in six isogenic clonal H9 hESC lines. Stem Cell Research, 45, 101779.
Döpper, Hannah; Horstmann, Marius; Menges, Julia; Bozet, Morgane; Kanber, Deniz & Steenpass, Laura
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Differentiation Protocol for 3D Retinal Organoids, Immunostaining and Signal Quantitation. Current Protocols in Stem Cell Biology, 55(1).
Döpper, Hannah; Menges, Julia; Bozet, Morgane; Brenzel, Alexandra; Lohmann, Dietmar; Steenpass, Laura & Kanber, Deniz
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RB1-Negative Retinal Organoids Display Proliferation of Cone Photoreceptors and Loss of Retinal Differentiation. Cancers, 14(9), 2166.
Kanber, Deniz; Woestefeld, Julia; Döpper, Hannah; Bozet, Morgane; Brenzel, Alexandra; Altmüller, Janine; Kilpert, Fabian; Lohmann, Dietmar; Pommerenke, Claudia & Steenpass, Laura
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Human stem cell models for retinoblastoma. PhD thesis.
Wöstefeld, J.
