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 a Lenka TESAŘOVÁ

Základní údaje

Originální název

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

Autoři

ŠIMARA, Pavel (203 Česká republika, domácí), Irena KRONTORÁD KOUTNÁ (203 Česká republika, garant, domácí), Stanislav STEJSKAL (203 Česká republika, domácí) a Lenka TESAŘOVÁ (203 Česká republika, domácí)

Vydání

ISSCR 2015 Annual Meeting, 2015

Další údaje

Jazyk

angličtina

Typ výsledku

Konferenční abstrakt

Obor

10601 Cell biology

Stát vydavatele

Švédsko

Utajení

není předmětem státního či obchodního tajemství

Odkazy

Poster abstract book ISSCR 2015

Kód RIV

RIV/00216224:14330/15:00080889

Organizační jednotka

Fakulta informatiky

Klíčová slova česky

hiPSC - reprogramming - genomic stability

Klíčová slova anglicky

hiPSC - reprogramming - genomic stability

Štítky

cbia-web

Změněno: 2. 3. 2018 10:00, Mgr. Pavel Šimara, Ph.D.

Anotace

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.

Návaznosti

CZ.1.07/2.3.00/30.0030, interní kód MU

Název: Rozvoj lidských zdrojů pro oblast buněčné biologie

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Rozvoj lidských zdrojů pro oblast buněčné biologie, 2.3 Lidské zdroje ve výzkumu a vývoji

GBP302/12/G157, projekt VaV

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

Investor: Grantová agentura ČR, Dynamika a organizace chromosomů během buněčného cyklu a při diferenciaci v normě a patologii

Citovat

ŠIMARA, Pavel, Irena KRONTORÁD KOUTNÁ, Stanislav STEJSKAL a 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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