Culture of human mesenchymal stem cells at low oxygen tension improves growth and genetic stability by activating glycolysis

Abstract

Expansion of human stem cells before cell therapy is typically performed at 20% O 2 . Growth in these pro-oxidative conditions can lead to oxidative stress and genetic instability. Here, we demonstrate that culture of human mesenchymal stem cells at lower, physiological O 2 concentrations significantly increases lifespan, limiting oxidative stress, DNA damage, telomere shortening and chromosomal aberrations. Our gene expression and bioenergetic data strongly suggest that growth at reduced oxygen tensions favors a natural metabolic state of increased glycolysis and reduced oxidative phosphorylation. We propose that this balance is disturbed at 20% O 2 , resulting in abnormally increased levels of oxidative stress. These observations indicate that bioenergetic pathways are intertwined with the control of lifespan and decisively influence the genetic stability of human primary stem cells. We conclude that stem cells for human therapy should be grown under low oxygen conditions to increase biosafety. Cell Death and Differentiation (2012) 19, 743-755; doi:10.1038/cdd.2011; published online 2 December 2011Human mesenchymal stem cells (hMSC) are being evaluated for the treatment of a large variety of pathologies, including traumatic lesions and cardiovascular and autoimmune diseases. 1,2 Although hMSC can be obtained from several tissues, they are scarce and their quantity and quality depends on a patient's clinical history, age, gender and genetic background. Most cell therapy protocols use 10-50 million hMSC per treatment, requiring expansion of extracted stem cells ex vivo for about 8 weeks before implantation. This expansion is typically performed under 'standard' non-physiological culture conditions, which among other factors expose cells to 20% O 2 , roughly 10 times the oxygen concentration encountered in their natural niches. 3,4 Previous studies have shown that exposure of mammalian cells to 20% O 2 concentrations induces DNA damage, thereby contributing decisively to cell senescence and loss of viability. [5][6][7] Conversely, culture of human stem cells over a physiological range of oxygen tensions (1-5%) improves cell growth, alters differentiation processes and extends lifespan. 8 Low oxygen tensions have also been shown to reduce the levels of double-strand breaks (DSB) and chromosomal abnormalities in several types of stem cells. 9,10 This evidence suggests that the poorly defined 'cell culture stress' can be a cause of genetic instability and therefore constitute a biological risk for cell therapy protocols. In agreement with this notion, we have found that short-term growth of hMSC at 20% O 2 significantly increases oxidative stress and DNA damage markers, DSB, chromosomal aberrations, aneuploidy and telomere shortening rates compared with cells grown at 3% O 2 . Despite these clear correlations, the mechanisms underlying the generation of genetic instability at high O 2 tension are mostly unknown.Mammalian cells have developed oxygen-sensing mechanisms to maintain cell and tissue homeostasis (reviewed...

Effect of low oxygen tension on the biological characteristics of human bone marrow mesenchymal stem cells

“…For instance, in a hypoxic environment, hypoxia-inducible factor 1α (HIF-1α) prevents TCA cycle activity and results in lower reactive oxygen species (ROS), slowing the rate of telomere shortening (Bodnar et al 1998;Richter and Zglinicki 2007); as a consequence, replicative senescence may be delayed. Moreover, a hypoxic environment induces higher proliferation rates (Estrada et al 2012;Fehrer et al 2007;Nekanti et al 2010) by lowering ROS levels and upregulating the expression of Notch target genes (e.g., Hes and Hey genes), resulting in the upregulation of several stem cell markers. For therapeutic applications, it will be important to improve the biological characteristics of stem cells under hypoxia to generate MSCs that can adapt to and function in the in vivo environment.…”

“…The ambient oxygen concentration might cause environmental stress to the in vitro cultured MSCs. Numerous studies have presented data correlating the ambient oxygen concentration with negative effects on MSCs, including early senescence, longer population doubling time, DNA damage (Estrada et al 2012;Fehrer et al 2007), and poor engraftment following transplantation (Mohamadnejad et al 2010;Schächinger et al 2006). These data have raised serious concerns regarding the therapeutic efficacy and safety of MSCs.…”

Long-term cultured mesenchymal stem cells frequently develop genomic mutations but do not undergo malignant transformation

“…Aneuploidy could be found not only at the late passage but also at early passages of in vitro culture. [9][10][11] Limited by the resolution of traditional karyotyping, it is not the most effective method for evaluation of genomic stability of MSCs for clinical applications. Some studies analyzed CNVs of in vitro-cultured BMMSCs and ADMSCs.…”

Asymmetric Aneuploidy in Mesenchymal Stromal Cells Detected by In Situ Karyotyping and Fluorescence In Situ Hybridization: Suggestions for Reference Values for Stem Cells

“…Previous studies reported that stem cells exhibited aneuploidy, which may be the result of a random process or a tendency of certain types of stem cells to develop abnormalities in specific chromosomes. In a previous study, four primary MSCs derived from adipose tissue were analyzed using FISH probes for chromosomes 8, 11, and 17, and these cells exhibited a substantially high percentage of aneuploidy, *15%-24%, even during the early passages of p2 to p5 [29]. In another study investigating induced pluripotent stem cells (iPSCs), the average aneuploidy rate was 2.1% for iPSCs and 4.2% for embryonic stem cells (ESCs) [30].…”