Pluripotent stem cells (PSCs) hold great promises for both disease modelling and regenerative medicine due to their potential to differentiate into cells of all three germ layers and their ability to self-renew and proliferate indefinitely in vitro. Their application, however, is hindered by their tendency to accumulate recurrent genetic variants during prolonged culture or reprogramming. For that reason, many recent studies focused on the optimization of culture conditions and passaging techniques to best preserve the cell’s genome. A crucial factor in genome stability is the availability of deoxynucleotide (dNTP) levels within a range that is optimal for DNA synthesis. Indeed, depleted or unbalanced dNTP pools are sources of replicative stress and genome instability. Despite that, dNTPs metabolism in PSCs is still an aspect that has been overlooked. Therefore, we studied the sizes of the DNA precursors in human induced pluripotent stem cells derived from keratinocytes (KiPSCs) in the two most used culture media, mTeSR-1 and E8. In particular, we investigated if nucleoside supplementation may adjust dNTP pool content and thus limiting replication stress. In order to detect and evaluate replicative stress in PSCs cultures we used as indicator γH2AX, immunodetected either by flow cytometry or by fluorescence microscopy. Experiments of nucleoside supplementation in PSCs culture showed an increase in the dNTP pool content in E8.

Investigating the correlation between culture conditions, deoxynucleotide metabolism and genomic instability in pluripotent stem cells

PAGLIARUSCO, TOMMASO
2021/2022

Abstract

Pluripotent stem cells (PSCs) hold great promises for both disease modelling and regenerative medicine due to their potential to differentiate into cells of all three germ layers and their ability to self-renew and proliferate indefinitely in vitro. Their application, however, is hindered by their tendency to accumulate recurrent genetic variants during prolonged culture or reprogramming. For that reason, many recent studies focused on the optimization of culture conditions and passaging techniques to best preserve the cell’s genome. A crucial factor in genome stability is the availability of deoxynucleotide (dNTP) levels within a range that is optimal for DNA synthesis. Indeed, depleted or unbalanced dNTP pools are sources of replicative stress and genome instability. Despite that, dNTPs metabolism in PSCs is still an aspect that has been overlooked. Therefore, we studied the sizes of the DNA precursors in human induced pluripotent stem cells derived from keratinocytes (KiPSCs) in the two most used culture media, mTeSR-1 and E8. In particular, we investigated if nucleoside supplementation may adjust dNTP pool content and thus limiting replication stress. In order to detect and evaluate replicative stress in PSCs cultures we used as indicator γH2AX, immunodetected either by flow cytometry or by fluorescence microscopy. Experiments of nucleoside supplementation in PSCs culture showed an increase in the dNTP pool content in E8.
2021
Investigating the correlation between culture conditions, deoxynucleotide metabolism and genomic instability in pluripotent stem cells
deoxynucleotides
stem cells
replicative stress
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.12608/42245