Hematopoietic and Stromal Stem Cells adapt themselves to hypoxia in culture which probably reflects their native hypoxic microenvironment [1–3]. Accordingly, several teams cultured HSC and MSC in hypoxic conditions in order to study their differentiation capacity [8–16, 19]. Another goal of these experiments is the hope of expanding these cells while maintaining their "stemness" properties. Although data from various laboratories are difficult to compare due to wide variations in oxygen tension, ranging from 0.1 to 5%, and the duration of culture, ranging from a few hours to 2 months, a few studies evidenced an early growth inhibition under hypoxia . Hypoxia induces cell cycle arrest in mammalian cells, however stem cells are more resistant to hypoxia than their progenies again reflecting their natural environment and their intrinsic quiescent state. We performed MSC cultures in 5% O2 which may be physiological for bone marrow stem cells . As MSC and HSC form a single bone marrow niche , 5% O2 tension is likely to be physiological for MSC as well. We observed that MSC grew slower under 5% O2 than under 21% O2 until P1, and gained a progressive growth advantage in the next passages, which matched previously published results . Meanwhile, hypoxic MSC expressed more adhesion and extracellular matrix molecules in early and late cultures, contained less mitochondria and displayed undifferentiated morphological features. In brief, early growth inhibition was somewhat expected and strikingly, GO analysis assigned down regulated genes to DNA metabolism and repair (POLQ, RRM2, XRCC2, FANCD2), cell cycle progression (E2F8, MKI67) and chromosomal organization (CENP-B, AURKB, KLF4) in agreement with our data on proliferation and colony size. Such inhibition likely contributes to the maintenance of MSC in a quiescent state, inasmuch as the inhibition of mitochondria may protect MSC from apoptosis. How could we reconcile these data with our observation that hypoxic MSC gained a growth advantage over normoxic MSC at late passages? The contradiction may be apparent. One possibility is that these cells became more sensitive to growth factors present in the serum. Whether growth advantage is due to a stimulation of proliferation pathways or to the expression of receptors for cytokines and growth factors or both, is worth investigating. Note in this respect that CXCR4 was induced by hypoxia.
As MSC in their niche are supposed to be quiescent and multipotent, these properties are apparently dissociated in our in vitro model, with quiescence being observed at early passages, whereas multipotency is augmented at late passages. Until we understand the in vivo signature of MSC, we cannot draw conclusions and pretend that in vitro culture in hypoxia mimics the niche.
Although expected from previous studies and suggested by our morphological observations, maintenance of stem cell characteristics at early passages under hypoxia was not inferred from GO analysis. Early induced genes were not assigned to multipotency but instead belonged mostly to adhesion molecules such as Von Willebrand Endothelial Cell Adhesion molecule and Protocadherin (Table 1). However, several genes may clearly affect stemness. CD93 regulates the clearance of apoptotic cells, a function critical to development, maintenance of homeostasis and tissue repair . The WNT-related transcription factor TCF1 may regulate MSC and enhance their osteogenic differentiation . At variance with the above genes, 8 genes potentially involved in the control of differentiation towards adipocytes, osteocytes and chondrocytes  were not modified by hypoxia [Additional file 1]. Strong expression of adhesion molecules may be physiologically relevant and correlate with broader differentiation potential of hypoxic MSC. Indeed VWF is a marker of endothelial commitment  and PLVAP, reported here for the first time in MSC is a leukocyte trafficking molecule  which may help transendothelial migration of MSC from the bone marrow. Stimulation of Leptin is also meaningful as a recent work demonstrated that it helps maintain mesenchymal progenitor cells undifferentiated . This result also shows that hypoxia impacts the metabolism of MSC in agreement with a study on rat MSC . In this study however, the duration of hypoxia was 24 hours only. Yet, several genes involved in adhesion and extracellular matrix were stimulated.
Hypoxia generates "plasticity". At P2 in hypoxia, only one group of genes was stimulated and was assigned to plasticity. SMOC2 is the first induced gene (Table 2) and plays a role in angiogenesis and extracellular matrix assembly , yet a recent article demonstrated that a related protein increases life span and fecundity in Drosophila . Kit gene was induced thus correlating with proliferation . LAMA1/laminin  and SNTG2/syntrophin gamma-2 [32, 33] are both involved in retinal and eye development whereas GPR56, a seven-transmembrane domain protein, is involved in brain cortical patterning .
We have observed that hypoxia stimulated several genes which converge to maintain the cells in an undifferentiated state, and facilitate transendothelial migration of MSC (Table 1 and 2). In parallel, hypoxia inhibited the expression of genes involved in cell proliferation (Table 1). This transcription profile probably reflects the intrinsic genetic program of MSC in vivo as these cells are quiescent, and endowed with migration and multilineage differentiation capacities. With respect to migration, note that CXCR4 was induced by hypoxia [Additional file 1 and reference 3] with potential implications in the egress of MSC from the bone marrow. This is in contrast with the cell surface phenotype of MSC which was almost unaffected in our experiments and in others . Note however that STRO-1 was expressed only transiently in cultured hypoxic but not in normoxic cells. This is not totally surprising since STRO-1 expression is gradually lost during culture expansion [18, 35]. Even though STRO-1 is useful to isolate MSC from various tissues, it is not positive on all MSC . Interestingly, STRO-1+ cells displayed enhanced expansion and multilineage differentiation potentialities [37, 38]. Thus, the expression of STRO-1 on hypoxic MSC may not be fortuitous and reflects multipotential status.
Our results may have physiological & medical applications. Oxygen tension is a critical parameter, possibly the most important one, in the culture of stem cells. As nestin-positive MSC and HSC form a unique bone marrow niche , hypoxia is undoubtedly a physiological milieu for MSC. In this respect it is worth mentioning that nestin was induced by hypoxia in our experiments [Additional file 1]. Given the ever growing therapeutic applications of MSC in regenerative medicine  and in autoimmune diseases , the impact of O2 on the functions of MSC should be carefully evaluated. For instance, intravenous injection of MSC results in their accumulation in the pulmonary parenchyma. Although this was sufficient to treat experimental septic shock , dissemination of MSC into other organs may be necessary to treat systemic diseases; induction of molecules involved in transendothelial migration as observed in our experiments may be helpful in this setting. Conversely however, hypoxia may be detrimental to other purposes. MSC inhibit TH17 cells in a CCL2-dependent manner by processing this chemokine to an antagonistic derivative, and may be helpful in the treatment of Experimental Allergic Enkephalitis (EAE) . Note in this respect that the transcription of CCL2 in MSC was inhibited under hypoxia in our experiments. Altogether our data demonstrate that hypoxia favoured the "undifferentiation program" of MSC, it remains to evaluate the impact of hypoxia on each desired function of these cells in the event of medical applications.
As the Holy Grail is to use tissue-specific cells derived from MSC in regenerative medicine, culture of MSC in hypoxia at least until P2 in order to induce the expression of a broad range of tissue-specific genes, may be beneficial, inasmuch as it also enhanced the cell numbers in parallel to their differentiating capacity. In this respect differentiation experiments should be carried to evaluate the potential of MSC to generate endothelial cells, myocytes and neurons. Finally, the most relevant result here is the demonstration of induction of plasticity, a major property of MSC, at variance with HSC .