DNA double strand breaks detection in pluripotent stem cells
with regard to cell cycle stages

a 2016

DNA double strand breaks detection in pluripotent stem cells with regard to cell cycle stages

ŠIMARA, Pavel, Irena KRONTORÁD KOUTNÁ, Lenka TESAŘOVÁ, Daniela ŘEHÁKOVÁ, Pavel MATULA et. al.

Basic information

Original name

DNA double strand breaks detection in pluripotent stem cells with regard to cell cycle stages

Authors

ŠIMARA, Pavel (203 Czech Republic, belonging to the institution), Irena KRONTORÁD KOUTNÁ (203 Czech Republic, guarantor, belonging to the institution), Lenka TESAŘOVÁ (203 Czech Republic, belonging to the institution), Daniela ŘEHÁKOVÁ (203 Czech Republic, belonging to the institution) and Pavel MATULA (203 Czech Republic, belonging to the institution)

Edition

ISSCR 2016 Annual Meeting, 2016

Other information

Language

English

Type of outcome

Konferenční abstrakt

Field of Study

10601 Cell biology

Country of publisher

United States of America

Confidentiality degree

není předmětem státního či obchodního tajemství

References:

Poster abstract book ISSCR 2016

RIV identification code

RIV/00216224:14330/16:00088039

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) have a potential in both disease modeling and regenerative medicine. It is of utmost importance that genomic integrity of the cells remains unharmed and DNA reparation systems fully functional. In our research we focus on the detection of DNA double strand breaks (DSBs) by phosphorylated histone H2AX (known as gammaH2AX) and p53-binding protein 1 (53BP1) in fibroblasts, three distinct lines of hiPSCs, and one line of human embryonic stem cells (hESCs). We measured both spontaneously occurred DSBs and DSBs induced by gamma-irradiation and its decrease in time. Foci number was detected by fluorescence microscopy and EdU (5-ethynyl-2-deoxyuridine) was used to discriminate between cell cycle stages. Discrimination between the EdU negative (G1) and positive (S/G2) populations allows excluding the replication-related foci and increase the accuracy of measurement. This is crucial when comparing number of DSBs in cell types with different cell cycle speed (ie. somatic cells and pluripotent cells). EdU discrimination is also valuable when the cell cycle is being modified during experiments in a way that changes proportion of cells in the S/G2 stage (ie. by irradiation or using cell cycle synchronising agents). In EdU negative (G1) group, we detected low number of replication non-related DSBs in fibroblasts, while this number increases significantly after reprogramming into hiPSCs to decrease again after long-term in vitro passaging. However, hiPSCs in high passages responded weakly to gamma-irradiation treatment in comparison to hiPSCs in low passage number, suggesting their DSB-reparation capacity may be compromised.

Links

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, Lenka TESAŘOVÁ, Daniela ŘEHÁKOVÁ and Pavel MATULA. DNA double strand breaks detection in pluripotent stem cells with regard to cell cycle stages. In ISSCR 2016 Annual Meeting. 2016.

@proceedings{1350040,
   author = {Šimara, Pavel and Krontorád Koutná, Irena and Tesařová, Lenka and Řeháková, Daniela and Matula, Pavel},
   booktitle = {ISSCR 2016 Annual Meeting},
   keywords = {hiPSC - reprogramming - genomic stability},
   language = {eng},
   title = {DNA double strand breaks detection in pluripotent stem cells with regard to cell cycle stages},
   url = {http://www.isscr.org/home/annual-meeting/san-francisco-2016/abstract-information},
   year = {2016}
}

TY  - CONF
ID  - 1350040
AU  - Šimara, Pavel - Krontorád Koutná, Irena - Tesařová, Lenka - Řeháková, Daniela - Matula, Pavel
PY  - 2016
TI  - DNA double strand breaks detection in pluripotent stem cells with regard to cell cycle stages
KW  - hiPSC - reprogramming - genomic stability
UR  - http://www.isscr.org/home/annual-meeting/san-francisco-2016/abstract-information
N2  - Human induced pluripotent stem cells (hiPSCs) have a potential in both disease modeling and regenerative medicine. It is of utmost importance that genomic integrity of the cells remains unharmed and DNA reparation systems fully functional. In our research we focus on the detection of DNA double strand breaks (DSBs) by phosphorylated histone H2AX (known as gammaH2AX) and p53-binding protein 1 (53BP1) in fibroblasts, three distinct lines of hiPSCs, and one line of human embryonic stem cells (hESCs). We measured both spontaneously occurred DSBs and DSBs induced by gamma-irradiation and its decrease in time. Foci number was detected by fluorescence microscopy and EdU (5-ethynyl-2-deoxyuridine) was used to discriminate between cell cycle stages. Discrimination between the EdU negative (G1) and positive (S/G2) populations allows excluding the replication-related foci and increase the accuracy of measurement. This is crucial when comparing number of DSBs in cell types with different cell cycle speed (ie. somatic cells and pluripotent cells). EdU discrimination is also valuable when the cell cycle is being modified during experiments in a way that changes proportion of cells in the S/G2 stage (ie. by irradiation or using cell cycle synchronising agents). In EdU negative (G1) group, we detected low number of replication non-related DSBs in fibroblasts, while this number increases significantly after reprogramming into hiPSCs to decrease again after long-term in vitro passaging. However, hiPSCs in high passages responded weakly to gamma-irradiation treatment in comparison to hiPSCs in low passage number, suggesting their DSB-reparation capacity may be compromised.
ER  -

Detailed Information on Publication Record