Cord blood culture in hypoxia: making the cells feel at home.

Until recently, cell and tissue culture protocols have emphasized the importance of pH control, adequate nutrients and growth factors, with little attention to oxygen (O 2 ) concentration in culture media. It is increasingly appreciated that adult tissues maintain localized domains where O 2 levels are much lower ( 1 – 9% O 2 ) than normal atmospheric conditions (21% O 2 ) commonly used in cell culture (1). Importantly, various stem cell types, including hematopoietic, mesenchymal and neural stem cells, are known to occupy these specialized hypoxic microenvironments or “ niches ” . In vitro studies employing low O 2 culture conditions ( 5% O 2 ) to mimic the hypoxic in vivo microenvironment have revealed a critical role of low O 2 tension to maintain undifferentiated stem cell phenotypes, and infl uence proliferation and cell-fate commitment (2). By promoting stem cell quiescence and offering a protective role against oxidative damage, hypoxia may be a strategic requirement to reduce the risk of mutagenesis in stem cell pools that serve as the source of differentiated cells and tissues for the life of the organism. Recent evidence of a convergence of pathways involved in hypoxia sensing and stem cell maintenance has emphasized the exciting possibility of stem cell regulation by hypoxia. The primary mediators of hypoxic adaptation are hypoxia-inducible factors (HIF), a family of transcription factors composed of two subunits, an oxygen-labile α subunit that rapidly stabilizes in response to low O 2 tensions (HIF1 α and HIF-2 α ), and a β subunit (HIF-1 β ) that is constitutively expressed (3). In immunoprecipitation assays, HIF-1 α physically interacted with the intracellular domain of Notch, a critical component for the maintenance of undifferentiated stem and progenitor cell populations, providing a striking molecular link between hypoxia and stemness (4). Similarly, HIF2 α was shown to be a direct upstream regulator of the transcriptional regulator of embryonic stem cell (ESC) pluripotency, Oct4, offering an additional mechanistic link to the emerging concept that low O 2 can modify stem cell function directly (5). WNT signaling is also well known to infl uence the properties of stem cells and, recently, HIF-1 α has been shown to maintain both adult and embryonic stem cells by directly regulating the WNT/ β -catenin pathway (6). In this issue of Cytotherapy , Buchheiser et al. (7) investigate the impact of hypoxia on umbilical cord blood (CB) stem cell populations. Low oxygen tension has been shown to enhance expansion of CB hematopoietic stem and progenitor cells compared to normoxic conditions (8). CB is also an attractive source of non-hematopoietic stem cells and, in normoxia, populations of mesenchymal stromal cells (MSC) (9), and unrestricted somatic stem cells (USSC) (10) have previously been identifi ed. CB MSC are defi ned as multipotent cells with osteogenic, chondrogenic, and adipogenic differentiation potential, as similarly reported in bone marrow (BM)-derived MSC. USSC were described in 2004 by this group as a multipotent cell population capable of differentiating into cells of all three germ layers both in vitro and in vivo (10). Unlike CB MSC, USSC do not express any of the HOX genes (11), perhaps pointing to different development origins, and do not exhibit adipogenic differentiation potential as correlated with a high expression of the adipogenesis inhibitor DLK1 (12). In this study (7), CB stem cell lines were initially derived in hypoxic and normoxic conditions, and further cultured for up to 100 days under both conditions. At fi rst, CB-H cells, the name coined to CB cell lines derived in hypoxia, appeared the same as cell lines derived in 20% O 2 (CB MSC, USSC); they were morphologically and immunophenotypically indistinguishable, and all cell types showed similar telomere length reduction with prolonged Cytotherapy, 2012; 14: 900–901