The effect of reprogramming method, source cell type and
long-term cell culture on genomic stability of human induced
pluripotent stem cells

a 2015

The effect of reprogramming method, source cell type and long-term cell culture on genomic stability of human induced pluripotent stem cells

ŠIMARA, Pavel, Irena KRONTORÁD KOUTNÁ, Stanislav STEJSKAL and Lenka TESAŘOVÁ

Basic information

Original name

The effect of reprogramming method, source cell type and long-term cell culture on genomic stability of human induced pluripotent stem cells

Authors

ŠIMARA, Pavel (203 Czech Republic, belonging to the institution), Irena KRONTORÁD KOUTNÁ (203 Czech Republic, guarantor, belonging to the institution), Stanislav STEJSKAL (203 Czech Republic, belonging to the institution) and Lenka TESAŘOVÁ (203 Czech Republic, belonging to the institution)

Edition

ISSCR 2015 Annual Meeting, 2015

Other information

Language

English

Type of outcome

Konferenční abstrakt

Field of Study

10601 Cell biology

Country of publisher

Sweden

Confidentiality degree

není předmětem státního či obchodního tajemství

References:

Poster abstract book ISSCR 2015

RIV identification code

RIV/00216224:14330/15:00080889

Organization unit

Faculty of Informatics

Keywords (in Czech)

hiPSC - reprogramming - genomic stability

Keywords in English

hiPSC - reprogramming - genomic stability

Abstract

V originále

Human induced pluripotent stem cells (hiPSCs) possess a great potential for clinical application. However, previous studies revealed the genomic instability of these cells. The reprogramming process itself may contribute to the mutational load in hiPSCs and subsequent in vitro culturing is also related to genomic aberrations increase. Various methods for iPSC generation were established and the main effort has been focused on reprogramming efficiency. In our study we try to figure out how reprogramming method, source cell type and long-term cell culture influences the genomic stability of hiPSCs. In our laboratory we established hiPSC clones from different source cells (fibroblasts or CD34+ blood progenitors) by three reprogramming methods: STEMCCA lentivirus, Sendai virus or episomal vectors. The pluripotency of our hiPSCs was verified by differentiation into all three germ layers and by teratoma assay. In order to study genomic integrity, we monitored DNA damage response (DDR). Phosphorylated form of histone H2AX (g-H2AX) and protein 53BP1 play key role in DDR mechanism and mark DNA lesions throughout the genome. The levels of g-H2AX and 53BP1 were determined in source cells, hiPSCs in low passage and hiPSCs in high passage. The immunofluorescence analysis revealed the differences in spontaneously occurring foci numbers among our hiPSC lines and variations were also found between low and high passages. Moreover, the samples differ in their capacity to response to ionizing radiation. Expectedly, the two proteins were extensively co-localized. We hypothesize that observed variations in DDR will correlate with the genomic aberrations, including duplications and deletions. Therefore, CGH microarray technology will be employed to detect copy number variations that may result from impaired DDR. The genomic stability is one of the major safety concerns of hiPSCs and must be addressed before transfer of this technology into clinical therapy.

Links

CZ.1.07/2.3.00/30.0030, interní kód MU

Name: Rozvoj lidských zdrojů pro oblast buněčné biologie

Investor: Ministry of Education, Youth and Sports of the CR, 2.3 Human resources in research and development

GBP302/12/G157, research and development project

Name: Dynamika a organizace chromosomů během buněčného cyklu a při diferenciaci v normě a patologii

Investor: Czech Science Foundation

Citovat

ŠIMARA, Pavel, Irena KRONTORÁD KOUTNÁ, Stanislav STEJSKAL and Lenka TESAŘOVÁ. The effect of reprogramming method, source cell type and long-term cell culture on genomic stability of human induced pluripotent stem cells. In ISSCR 2015 Annual Meeting. 2015.

@proceedings{1305881,
   author = {Šimara, Pavel and Krontorád Koutná, Irena and Stejskal, Stanislav and Tesařová, Lenka},
   booktitle = {ISSCR 2015 Annual Meeting},
   keywords = {hiPSC - reprogramming - genomic stability},
   language = {eng},
   title = {The effect of reprogramming method, source cell type and long-term cell culture on genomic stability of human induced pluripotent stem cells},
   url = {http://www.isscr.org/home/annual-meeting/past-future-meetings/isscr2015},
   year = {2015}
}

TY  - CONF
ID  - 1305881
AU  - Šimara, Pavel - Krontorád Koutná, Irena - Stejskal, Stanislav - Tesařová, Lenka
PY  - 2015
TI  - The effect of reprogramming method, source cell type and long-term cell culture on genomic stability of human induced pluripotent stem cells
KW  - hiPSC - reprogramming - genomic stability
UR  - http://www.isscr.org/home/annual-meeting/past-future-meetings/isscr2015
L2  - http://www.isscr.org/home/annual-meeting/past-future-meetings/isscr2015
N2  - Human induced pluripotent stem cells (hiPSCs) possess a great potential for clinical application. However, previous studies revealed the genomic instability of these cells. The reprogramming process itself may contribute to the mutational load in hiPSCs and subsequent in vitro culturing is also related to genomic aberrations increase. Various methods for iPSC generation were established and the main effort has been focused on reprogramming efficiency. In our study we try to figure out how reprogramming method, source cell type and long-term cell culture influences the genomic stability of hiPSCs. In our laboratory we established hiPSC clones from different source cells (fibroblasts or CD34+ blood progenitors) by three reprogramming methods: STEMCCA lentivirus, Sendai virus or episomal vectors. The pluripotency of our hiPSCs was verified by differentiation into all three germ layers and by teratoma assay. In order to study genomic integrity, we monitored DNA damage response (DDR). Phosphorylated form of histone H2AX (g-H2AX) and protein 53BP1 play key role in DDR mechanism and mark DNA lesions throughout the genome. The levels of g-H2AX and 53BP1 were determined in source cells, hiPSCs in low passage and hiPSCs in high passage. The immunofluorescence analysis revealed the differences in spontaneously occurring foci numbers among our hiPSC lines and variations were also found between low and high passages. Moreover, the samples differ in their capacity to response to ionizing radiation. Expectedly, the two proteins were extensively co-localized. We hypothesize that observed variations in DDR will correlate with the genomic aberrations, including duplications and deletions. Therefore, CGH microarray technology will be employed to detect copy number variations that may result from impaired DDR. The genomic stability is one of the major safety concerns of hiPSCs and must be addressed before transfer of this technology into clinical therapy.
ER  -

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