DNA double-strand breaks in human induced pluripotent stem cell
reprogramming and long-term in vitro culturing

J 2017

DNA double-strand breaks in human induced pluripotent stem cell reprogramming and long-term in vitro culturing

ŠIMARA, Pavel, Lenka TESAŘOVÁ, Daniela ŘEHÁKOVÁ, Pavel MATULA, Stanislav STEJSKAL et. al.

Basic information

Original name

DNA double-strand breaks in human induced pluripotent stem cell reprogramming and long-term in vitro culturing

Authors

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

Edition

Stem Cell Research & Therapy, Springer Nature, 2017, 1757-6512

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

10601 Cell biology

Country of publisher

Germany

Confidentiality degree

není předmětem státního či obchodního tajemství

References:

URL

Impact factor

Impact factor: 4.963

RIV identification code

RIV/00216224:14330/17:00094705

Organization unit

Faculty of Informatics

DOI

http://dx.doi.org/10.1186/s13287-017-0522-5

UT WoS

000396978500002

Keywords in English

Human induced pluripotent stem cells; DNA double-strand breaks; gammaH2AX; 53BP1; Long-term in vitro culture; DNA repair

Abstract

V originále

BACKGROUND: Human induced pluripotent stem cells (hiPSCs) play roles in both disease modelling and regenerative medicine. It is critical that the genomic integrity of the cells remains intact and that the DNA repair systems are fully functional. In this article, we focused on the detection of DNA double-strand breaks (DSBs) by phosphorylated histone H2AX (known as gammaH2AX) and p53-binding protein 1 (53BP1) in three distinct lines of hiPSCs, their source cells, and one line of human embryonic stem cells (hESCs). METHODS: We measured spontaneously occurring DSBs throughout the process of fibroblast reprogramming and during long-term in vitro culturing. To assess the variations in the functionality of the DNA repair system among the samples, the number of DSBs induced by gamma-irradiation and the decrease over time was analysed. The foci number was detected by fluorescence microscopy separately for the G1 and S/G2 cell cycle phases. RESULTS: We demonstrated that fibroblasts contained a low number of non-replication-related DSBs, while this number increased after reprogramming into hiPSCs and then decreased again after long-term in vitro passaging. The artificial induction of DSBs revealed that the repair mechanisms function well in the source cells and hiPSCs at low passages, but fail to recognize a substantial proportion of DSBs at high passages. CONCLUSIONS: Our observations suggest that cellular reprogramming increases the DSB number but that the repair mechanism functions well. However, after prolonged in vitro culturing of hiPSCs, the repair capacity decreases.

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

LM2015090, research and development project

Name: Český národní uzel Evropské sítě infrastruktur klinického výzkumu (Acronym: CZECRIN)

Investor: Ministry of Education, Youth and Sports of the CR

NV16-31501A, research and development project

Name: Tkáňové inženýrství epitelů: Buňky a protokoly pro regenerativní medicínu

Citovat

ŠIMARA, Pavel, Lenka TESAŘOVÁ, Daniela ŘEHÁKOVÁ, Pavel MATULA, Stanislav STEJSKAL, Aleš HAMPL and Irena KRONTORÁD KOUTNÁ. DNA double-strand breaks in human induced pluripotent stem cell reprogramming and long-term in vitro culturing. Stem Cell Research & Therapy. Springer Nature, 2017, vol. 8, No 73, p. 1-13. ISSN 1757-6512. Available from: https://dx.doi.org/10.1186/s13287-017-0522-5.

@article{1377715,
   author = {Šimara, Pavel and Tesařová, Lenka and Řeháková, Daniela and Matula, Pavel and Stejskal, Stanislav and Hampl, Aleš and Krontorád Koutná, Irena},
   article_number = {73},
   doi = {http://dx.doi.org/10.1186/s13287-017-0522-5},
   keywords = {Human induced pluripotent stem cells; DNA double-strand breaks; gammaH2AX; 53BP1; Long-term in vitro culture; DNA repair},
   language = {eng},
   issn = {1757-6512},
   journal = {Stem Cell Research & Therapy},
   title = {DNA double-strand breaks in human induced pluripotent stem cell reprogramming and long-term in vitro culturing},
   url = {https://stemcellres.biomedcentral.com/articles/10.1186/s13287-017-0522-5},
   volume = {8},
   year = {2017}
}

TY  - JOUR
ID  - 1377715
AU  - Šimara, Pavel - Tesařová, Lenka - Řeháková, Daniela - Matula, Pavel - Stejskal, Stanislav - Hampl, Aleš - Krontorád Koutná, Irena
PY  - 2017
TI  - DNA double-strand breaks in human induced pluripotent stem cell reprogramming and long-term in vitro culturing
JF  - Stem Cell Research & Therapy
VL  - 8
IS  - 73
SP  - 1-13
EP  - 1-13
PB  - Springer Nature
SN  - 17576512
KW  - Human induced pluripotent stem cells
KW  - DNA double-strand breaks
KW  - gammaH2AX
KW  - 53BP1
KW  - Long-term in vitro culture
KW  - DNA repair
UR  - https://stemcellres.biomedcentral.com/articles/10.1186/s13287-017-0522-5
L2  - https://stemcellres.biomedcentral.com/articles/10.1186/s13287-017-0522-5
N2  - BACKGROUND: Human induced pluripotent stem cells (hiPSCs) play roles in both disease modelling and regenerative medicine. It is critical that the genomic integrity of the cells remains intact and that the DNA repair systems are fully functional. In this article, we focused on the detection of DNA double-strand breaks (DSBs) by phosphorylated histone H2AX (known as gammaH2AX) and p53-binding protein 1 (53BP1) in three distinct lines of hiPSCs, their source cells, and one line of human embryonic stem cells (hESCs). METHODS: We measured spontaneously occurring DSBs throughout the process of fibroblast reprogramming and during long-term in vitro culturing. To assess the variations in the functionality of the DNA repair system among the samples, the number of DSBs induced by gamma-irradiation and the decrease over time was analysed. The foci number was detected by fluorescence microscopy separately for the G1 and S/G2 cell cycle phases. RESULTS: We demonstrated that fibroblasts contained a low number of non-replication-related DSBs, while this number increased after reprogramming into hiPSCs and then decreased again after long-term in vitro passaging. The artificial induction of DSBs revealed that the repair mechanisms function well in the source cells and hiPSCs at low passages, but fail to recognize a substantial proportion of DSBs at high passages. CONCLUSIONS: Our observations suggest that cellular reprogramming increases the DSB number but that the repair mechanism functions well. However, after prolonged in vitro culturing of hiPSCs, the repair capacity decreases.
ER  -

ŠIMARA, Pavel, Lenka TESAŘOVÁ, Daniela ŘEHÁKOVÁ, Pavel MATULA, Stanislav STEJSKAL, Aleš HAMPL and Irena KRONTORÁD KOUTNÁ. DNA double-strand breaks in human induced pluripotent stem cell reprogramming and long-term in vitro culturing. \textit{Stem Cell Research \&{} Therapy}. Springer Nature, 2017, vol.~8, No~73, p.~1-13. ISSN~1757-6512. Available from: https://dx.doi.org/10.1186/s13287-017-0522-5.

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