Human Adipose-Derived Stem Cells Expanded Under Ambient Oxygen Concentration Accumulate Oxidative DNA Lesions and Experience Procarcinogenic DNA Replication Stress

Rémy Bétous (1, 2, 3) , Marie-Laure Renoud (3, 4, 5) , Claire Hoede (6, 7) , Ignacio González (6, 7) , Natalie Jones (8) , Michel Longy (8) , Luc Sensebé (3, 4, 5) , Christophe Cazaux (1, 2, 3) , Jean-Sébastien Hoffmann (1, 2, 3)

1 Equipe labellisée Ligue contre le Cancer
2 CRCT - Centre de Recherches en Cancérologie de Toulouse
3 UT3 - Université Toulouse III - Paul Sabatier
4 Etablissement Français du Sang Pyrénées-Méditerranée
5 STROMALab
6 MIAT INRA - Unité de Mathématiques et Informatique Appliquées de Toulouse
7 Plateforme Bio-Informatique - Génotoul
8 Institut Bergonié [Bordeaux]

Adipose-derived stem cells (ADSCs) have led to growing interest in cell-based therapy because they can be easily harvested from an abundant tissue. ADSCs must be expanded in vitro before transplantation. This essential step causes concerns about the safety of adult stem cells in terms of potential transformation. Tumorigenesis is driven in its earliest step by DNA replication stress, which is characterized by the accumulation of stalled DNA replication forks and activation of the DNA damage response. Thus, to evaluate the safety of ADSCs during ex vivo expansion, we monitored DNA replication under atmospheric (21%) or physiologic (1%) oxygen concentration. Here, by combining immunofluorescence and DNA combing, we show that ADSCs cultured under 21% oxygen accumulate endogenous oxidative DNA lesions, which interfere with DNA replication by increasing fork stalling events, thereby leading to incomplete DNA replication and fork collapse. Moreover, we found by RNA sequencing (RNA-seq) that culture of ADSCs under atmospheric oxygen concentration leads to misexpression of cell cycle and DNA replication genes, which could contribute to DNA replication stress. Finally, analysis of acquired small nucleotide polymorphism shows that expansion of ADSCs under 21% oxygen induces a mutational bias toward deleterious transversions. Overall, our results suggest that expanding ADSCs at a low oxygen concentration could reduce the risk for DNA replication stress-associated transformation, as occurs in neoplastic tissues. SIGNIFICANCE:The present work explored the influence of oxygen concentration on the induction of DNA replication stress, a major driver of tumorigenesis, during ex vivo amplification of adipose-derived stem cells (ADSCs) used for cellular therapies. The currently used good manufacturing practices atmospheric oxygen condition (21%) compared with the physiologic one (1%) increased oxidative DNA damage and perturbed DNA replication by blocking DNA replication forks and, as a result, enhanced transversion mutations and activated the DNA damage response pathways. Collectively, these results strongly suggest that decreasing oxygen concentration could improve the safety of ADSCs during ex vivo expansion.

Mots clés: DNA damage; DNA replication stress; Ex vivo expansion; Human adipose-derived stem cells; Oxygen concentration

Domaines: Sciences du Vivant [q-bio]

Rémy Bétous, Marie-Laure Renoud, Claire Hoede, Ignacio González, Natalie Jones, et al.. Human Adipose-Derived Stem Cells Expanded Under Ambient Oxygen Concentration Accumulate Oxidative DNA Lesions and Experience Procarcinogenic DNA Replication Stress. Stem Cells Translational Medicine, 2016, 6 (1), pp.68-76. ⟨10.5966/sctm.2015-0401⟩. ⟨hal-01604404⟩