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Transcriptome Profiling Reveals Degree of Variability in Induced Pluripotent Stem Cell Lines: Impact for Human Disease Modeling
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik.ORCID iD: 0000-0002-4383-9880
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.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.ORCID iD: 0000-0001-6085-6749
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2015 (English)In: Cellular Reprogramming, ISSN 2152-4971, E-ISSN 2152-4998, Vol. 17, no 5, 327-337 p.Article in journal (Refereed) Published
Abstract [en]

Induced pluripotent stem cell (iPSC) technology has become an important tool for disease modeling. Insufficient data on the variability among iPSC lines derived from a single somatic parental cell line have in practice led to generation and analysis of several, usually three, iPSC sister lines from each parental cell line. We established iPSC lines from a human fibroblast line (HDF-K1) and used transcriptome sequencing to investigate the variation among three sister lines (iPSC-K1A, B, and C). For comparison, we analyzed the transcriptome of an iPSC line (iPSC-K5B) derived from a different fibroblast line (HDF-K5), a human embryonic stem cell (ESC) line (ESC-HS181), as well as the two parental fibroblast lines. All iPSC lines fulfilled stringent criteria for pluripotency. In an unbiased cluster analysis, all stem cell lines (four iPSCs and one ESC) clustered together as opposed to the parental fibroblasts. The transcriptome profiles of the three iPSC sister lines were indistinguishable from each other, and functional pathway analysis did not reveal any significant hits. In contrast, the expression profiles of the ESC line and the iPSC-K5B line were distinct from that of the sister lines iPSC-K1A, B, and C. Differentiation to embryoid bodies and subsequent analysis of germ layer markers in the five stem cell clones confirmed that the distribution of their expression profiles was retained. Taken together, our observations stress the importance of using iPSCs of different parental origin rather than several sister iPSC lines to distinguish disease-associated mechanisms from genetic background effects in disease modeling.

Place, publisher, year, edition, pages
2015. Vol. 17, no 5, 327-337 p.
National Category
Other Biological Topics Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:uu:diva-244422DOI: 10.1089/cell.2015.0009ISI: 000361523600002PubMedID: 26348590OAI: oai:DiVA.org:uu-244422DiVA: diva2:792379
Funder
Swedish Research Council, K2013-66X-10829-20-3 621-2009-4629EU, European Research Council, 282330AstraZenecaScience for Life Laboratory - a national resource center for high-throughput molecular bioscienceSwedish National Infrastructure for Computing (SNIC), b2013214
Available from: 2015-03-03 Created: 2015-02-16 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)
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Available from: 2015-04-01 Created: 2015-03-03 Last updated: 2015-09-24

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Schuster, JensHalvardson, JonatanLorenzo, Laureanne PilarAmeur, AdamSobol, MariaRaykova, DoroteyaAnnerén, GöranFeuk, LarsDahl, Niklas

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Medicinsk genetik och genomikDepartment of Immunology, Genetics and Pathology
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Cellular Reprogramming
Other Biological TopicsMedical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)

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