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Methods of Reprogramming to Induced Pluripotent Stem Cell Associated with Chromosomal Integrity and Delineation of a Chromosome 5q Candidate Region for Growth Advantage
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik.ORCID iD: 0000-0001-6452-2199
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik.
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2015 (English)In: Stem Cells and Development, ISSN 1547-3287, E-ISSN 1557-8534, Vol. 24, no 17, 2032-2040 p.Article in journal (Refereed) Published
Abstract [en]

Induced pluripotent stem cells (iPSCs) have brought great promises for disease modeling and cell-based therapies. One concern related to the use of reprogrammed somatic cells is the loss of genomic integrity and chromosome stability, a hallmark for cancer and many other human disorders. We investigated 16 human iPSC lines reprogrammed by nonintegrative Sendai virus (SeV) and another 16 iPSC lines generated by integrative lentivirus for genetic changes. At early passages we detected cytogenetic rearrangements in 44% (7/16) of iPSC lines generated by lentiviral integration whereas the corresponding figure was 6% (1/16) using SeV-based delivery. The rearrangements were numerical and/or structural with chromosomes 5 and 12 as the most frequently involved chromosomes. Three iPSC lines with chromosome 5 aberrations were derived from one and the same donor. We present in this study the aberrant karyotypes including a duplication of chromosome 5q13q33 that restricts a candidate region for growth advantage. Our results suggest that the use of integrative lentivirus confers a higher risk for cytogenetic abnormalities at early passages when compared to SeV-based reprogramming. In combination, our findings expand the knowledge on acquired cytogenetic aberrations in iPSC after reprogramming and during culture.

Place, publisher, year, edition, pages
2015. Vol. 24, no 17, 2032-2040 p.
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biology with specialization in Molecular Cell Biology
Identifiers
URN: urn:nbn:se:uu:diva-246225DOI: 10.1089/scd.2015.0061ISI: 000359606100007OAI: oai:DiVA.org:uu-246225DiVA: diva2:792386
Available from: 2015-03-03 Created: 2015-03-03 Last updated: 2017-12-04Bibliographically approved
In thesis
1. Genetics of Two Mendelian Traits and Validation of Induced Pluripotent Stem Cell (iPSC) Technology for Disease Modeling
Open this publication in new window or tab >>Genetics of Two Mendelian Traits and Validation of Induced Pluripotent Stem Cell (iPSC) Technology for Disease Modeling
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Novel technologies for genome analysis have provided almost unlimited opportunities to uncover structural gene variants behind human disorders. Whole exome sequencing (WES) is especially useful for understanding rare Mendelian conditions, because it reduces the requirements for a priori clinical data, and can be applied on a small number of patients. However, supporting functional data on the effect of specific gene variants are often required to power these findings. A variety of methods and biological model systems exists for this purpose. Among those, induced pluripotent stem cells (iPSCs), which are capable of self-renewal and differentiation, stand out as an alternative to animal models.

In papers I and II we took advantage of WES to identify gene variants underlying autosomal recessive pure hair and nail ectodermal dysplasia (AR PHNED) as well as autosomal dominant familial visceral myopathy (FVM). We identified a homozygous variant c.821T>C (p.Phe274Ser) in the KRT74 gene as the causative mutation in AR PHNED, supported by the fact that Keratin-74 was undetectable in hair follicles of an affected family member. In a family segregating FVM we found a heterozygous tandem base substitution c.806_807delinsAA (p.(Gly269Glu)) in the ACTG2 gene in the affected members. This novel variant is associated with a broad range of visceral symptoms and a variable age of onset.

In Paper III we explored the similarity between clonally derived iPSC lines originating from a single parental fibroblast line and we highlighted the necessity to use lines originating from various donors in disease modeling because of biological variation. Paper IV focused on how the genomic integrity of iPSCs is affected by the choice of reprogramming methods. We described several novel cytogenetic rearrangements in iPSCs and we identified a chromosome 5q duplication as a candidate aberration for growth advantage.

In summary, this doctoral thesis brings novel findings on unreported disease-causing variants, as supported by extensive genetic analysis and functional data. A novel molecular mechanism behind AR PHNED is presented and the phenotypic spectrum associated with FVM is expanded. In addition, the thesis brings novel understanding of benefits and limitations of the iPSC technology to be considered for disease modeling.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. 54 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1078
Keyword
Disease modeling, Mendelian disorders, iPSC, Whole exome sequencing, Transcriptome sequencing
National Category
Genetics Cell Biology Medical Genetics
Research subject
Medical Science
Identifiers
urn:nbn:se:uu:diva-246228 (URN)978-91-554-9184-0 (ISBN)
Public defence
2015-04-24, Fåhraeussalen, Rudbeck Laboratoriet, Dag Hammarsjöldsväg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2015-04-01 Created: 2015-03-03 Last updated: 2015-09-24

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Sobol, MariaRaykova, DoroteyaCavelier, LuciaKhalfallah, AydaSchuster, JensDahl, Niklas

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