ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Endocrine Abstracts (2017) 50 OC6.3 | DOI: 10.1530/endoabs.50.OC6.3

Generation of multiple endocrine neoplasia type 1 and death-domain-associated protein pluripotent stem cell lines to investigate mechanisms of pancreatic neuroendocrine tumourigenesis

Kumara Dissanayake, Lindsay Davidson, Conor Poland & Paul Newey

University of Dundee, Dundee, UK.

Background: Despite a wealth of gene-discovery studies identifying recurrently mutated genes in hereditary and sporadic endocrine tumours, the molecular mechanisms underpinning tumourigenesis frequently remain ill-defined, in part reflecting a lack of physiologically relevant model systems to investigate gene function. Here, using pancreatic neuroendocrine tumours as an example, we explored the utility of human induced pluripotent stem cell (iPSCs) and CRISPR/Cas9 gene-editing to investigate how inactivating mutations in MEN1 and DAXX contribute to tumour formation.

Methods: 20-nucleotide guide RNAs (gRNAs) were designed to target the coding-region of MEN1 (exons 2/3) and DAXX (exon 3), and were cloned into a bicistronic Cas9/scaffold-RNA vector (with puromycin resistance selection cassette). The targeting efficiency of each gRNAs was evaluated following transfection in HEK293 and/or HeLa cells. ChIPS4 iPSCs were subsequently electroporated with validated MEN1 or DAXX gRNA/Cas9 vectors and single-cell clonal populations established. Pluripotency markers were visualised by immunofluorescence, whilst MEN1 and DAXX gene-targeting was assessed by western blot and DNA sequence analysis.

Results: gRNAs targeting MEN1 and DAXX were functionally active in HEK293 and HeLa cells, resulting in mono-allelic or bi-allelic inactivating mutations at the respective genomic sites. Similar activity was observed in iPSCs with the generation of 6 mutant MEN1 lines (3 mono-allelic, 3 bi-allelic) and 6 mutant DAXX lines (5 mono-allelic, 1 bi-allelic). When compared with wild-type iPSCs, MEN1 mutant lines demonstrated no initial differences in cell morphology or pluripotency marker expression. However, after serial passage, colonies with bi-allelic MEN1 inactivation underwent spontaneous differentiation with reduced proliferation. Similarly, although mono-allelic DAXX mutant cells were indistinguishable from wild-type iPSCs, bi-allelic DAXX inactivation resulted in cellular differentiation, reduced proliferation and a phenotype consistent with senescence.

Conclusions: CRISPR/Cas9 gene-editing provides an efficient tool to generate genetically-tractable stem cell models to investigate endocrine tumourigenesis. Here, we demonstrate that expression of Menin and DAXX is likely required for long-term maintenance of pluripotency.

Article tools

My recent searches

No recent searches.