Functional analysis of a chromosomal deletion associated with myelodysplastic syndromes using isogenic human induced pluripotent stem cells

Developers

Andriana G Kotini, Chan-Jung Chang, Stephen D Nimer, Eirini P Papapetrou, etc.

Description of the technology

Chromosomal deletions associated with human diseases, such as cancer, are common, but synteny (i.e. structural similarity of gene linkage groups in organisms of different species) complicates modeling of these deletions in mice.

To solve the synteny issues the authors of the technology use cellular reprogramming and genome engineering to functionally dissect the loss of chromosome 7q (del(7q)), a somatic cytogenetic abnormality present in myelodysplastic syndromes (MDS). Isogenic karyotypically normal induced pluripotent stem cells (iPSCs) and iPSCs with del(7q) were derived from hematopoietic cells of MDS patients. It was showed with the help of these cells that the del(7q) iPSCs recapitulate disease-associated phenotypes, including impaired hematopoietic differentiation. These disease phenotypes were rescued by spontaneous gene dosage correction. They could be reproduced in karyotypically normal cells by engineering hemizygosity of defined chr7q segments in a 20-Mb region. We use a phenotype-rescue screen to identify candidate haploinsufficient genes that might mediate the del(7q)-hematopoietic defect.

In general, a proposed workflow was implemented by this way: i) generation of isogenic hPSCs harboring the specific deletion; ii) determination of a phenotype in a disease-relevant cell type derived from hPSCs; iii) identification of candidate haploinsufficient genes based on their differential expression in the diploid vs haploid state and iv) filtration of them through phenotype-rescue screens to select one or few candidates for follow-up studies.

This dissecting the MDS-iPSC phenotypes at the molecular level could provide understanding of the pathogenesis of myelodysplasia and leukemic transformation.

Practical application

The technology is a strategy for functional cancer genetics that should prove applicable to the study of disease-associated chromosomal deletions. This strategy is carried out as a gain-of-function (rescue) assay, which provides substantial advantages over knockdown/knockout screens. It is a generalizeable approach to the discovery of haploinsufficient genes. Because a gene can be mono-allelically inactivated in different ways, this approach can be integrated with data on different classes of mutation (copy number variants, single nucleotide polymorphisms) from cancer genome databases to inform gene prioritization.

This approach highlights the utility of human iPSCs both for functional mapping of disease-associated large-scale chromosomal deletions and for discovery of haploinsufficient genes.

Besides, the robustness of cellular MDS phenotypes, obtained with this technology, can provide a platform for phenotype-driven drug screens to identify small molecules that can cure these pathological phenotypes.

Laboratories

• Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York (USA)
• The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York (USA)
• The Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York (USA)
• Sylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Miami, Florida, USA.
• Division of Hematology, Department of Medicine, University of Washington, Seattle (USA)
• Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle (USA)
• Department of Pathology, University of Washington, Seattle (USA)

Links

Publications

• Kotini, A.G. et al. «Functional analysis of a chromosomal deletion associated with myelodysplastic syndromes using isogenic human induced pluripotent stem cells." 33.6 Nat Biotechnol. (2015): 646−655.
• Papapetrou, E.P. et al. «Genomic safe harbors permit high beta-globin transgene expression in thalassemia induced pluripotent stem cells." 29 Nat. Biotechnol. (2011): 73–78.
• Papapetrou, E.P. & Sadelain, M. «Generation of transgene-free human induced pluripotent stem cells with an excisable single polycistronic vector." 6 Nat. Protoc. (2011): 1251–1273.