Hypoxia Differentially Modulates the Genomic Stability of Clinical-Grade ADSCs and BM-MSCs in Long-Term Culture

Abstract

Long-term cultures under hypoxic conditions have been demonstrated to maintain the phenotype of mesenchymal stromal/stem cells (MSCs) and to prevent the emergence of senescence. According to several studies, hypoxia has frequently been reported to drive genomic instability in cancer cells and in MSCs by hindering the DNA damage response and DNA repair. Thus, we evaluated the occurrence of DNA damage and repair events during the ex vivo expansion of clinical-grade adipose-derived stromal cells (ADSCs) and bone marrow (BM)-derived MSCs cultured with platelet lysate under 21% (normoxia) or 1% (hypoxia) O 2 conditions. Hypoxia did not impair cell survival after DNA damage, regardless of MSC origin. However, ADSCs, unlike BMMSCs, displayed altered cH2AX signaling and increased ubiquitylated cH2AX levels under hypoxic conditions, indicating an impaired resolution of DNA damage-induced foci. Moreover, hypoxia specifically promoted BM-MSC DNA integrity, with increased Ku80, TP53BP1, BRCA1, and RAD51 expression levels and more efficient nonhomologous end joining and homologous recombination repair. We further observed that hypoxia favored mtDNA stability and maintenance of differentiation potential after genotoxic stress. We conclude that long-term cultures under 1% O 2 were more suitable for BM-MSCs as suggested by improved genomic stability compared with ADSCs. STEM CELLS 2015;33:3608-3620 SIGNIFICANCE STATEMENTHuman mesenchymal stem cells (MSCs) feature differentiation capacities, immunomodulatory properties and therefore represent a great potential for medicine. In vitro culture allows their amplification for suitable clinical uses. Hypoxia seems relevant for the prevention of senescence and loss of MSC features. However, hypoxia is shown to promote cancer or DNA damage response (DDR) and repair failures. We demonstrate that long-term culture of MSCs with platelet lysate in hypoxia does not necessarily inhibit DDR and DNA repair. Here, bone marrowMSCs conserved or rather improved their DDR and DNA repair (NHEJ, HR), mtDNA stability in hypoxia compared to adipose-derived stem cells. Moreover, the ubiquitylation status of gH2AX between BM-MSCs and ADSCs might be responsible for changes in their DNA repair activities. Appropriate oxygen tension seems crucial to preserve the MSCs genomic stability according to their origins.

Evaluation of Three Devices for the Isolation of the Stromal Vascular Fraction from Adipose Tissue and for ASC Culture: A Comparative Study

“…Transformation process is a complicated sequence of events which might occur when the cells are grown in vitro [45, 46]. Nowadays, there is no evidence of a real genetic instability of ASC and MSC in general [47], but a small number of publications highlighted few aberrations [48, 49], and some publications [50, 51] have been retracted due to cell culture contamination [52, 53]. Moreover, we deeply think that closed system that strikingly limit the microbial contamination allow surgical unit and GMP-laboratories to preserve the safety of the patient.…”

Should hypoxia preconditioning become the standardized procedure for bone marrow MSCs preparation for clinical use?

“…In conclusion, the study of Bigot and colleagues [10] is relevant not only for showing the genomic stability of BM-MSCs under hypoxia, but also raises an essential issue for further discussion and investigations to consider the possibility of adding hypoxia preconditioning to the standardized protocol of MSCs expansion for clinical use. Hypoxia preconditioning is beneficial to some extent, but further investigations are required to standardize the protocol by basic scientific researchers and clinicians; importantly, a gathering of experts led by regulatory agencies, academic societies or scientific journals in the field of MSC-based therapy is encouraged to optimize the use of hypoxia preconditioning for MSCs expansion, as MSC-based therapy in clinics is increasing significantly worldwide.…”

“…In a recent paper published in this journal by Bigot and coauthors, hypoxia was reported to preserve the genomic stability of BM-MSCs after long-term culture in platelet lysate (PL) [10]. The authors found that mimicking the niche microenvironment with hypoxia could promote the DNA damage response, DNA repair and mitochondrial DNA stability of BM-MSCs cultured with PL; in comparison, adipose-derived stromal cells (ADSCs) cultured in the same conditions possessed lower DNA damage response and DNA repair ability.…”

Comparative treatment efficiency of adipose and bone marrow derived allogenic mesenchymal stem cell transplantation in mouse models of liver fibrosis

“…Both in vitro and in vivo studies have suggested differences in differentiation capacity and genetic profile amongst populations of cells, regardless of similarities in cell morphology, surface markers or differentiation potential (Reinisch et al, 2015). Studies by Bigot et al (2015) have also indicated that the oxygen levels can affect the stability of the genetic profiles of AD-MSCs and BM-MSCs in different ways (Bigot et al, 2015). These phenomena might account for variations in the efficiency of MSCs acquired from various sources for therapeutic applications.…”