In this paper, we provide the evidence for higher in vitro targeted migration of hMSC cultured under hypoxic conditions compared to the cells grown in normal oxygen toward most of the tested cytokines. Previous studies showed the enhancing effect of short term exposure to hypoxia on migration of MSC [15, 19, 28, 32–34]; an increase in cell velocity and Euclidian distance on a series of matrices for the hMSC cultured in 2% hypoxia has also been shown . To our knowledge we are the first to report higher targeted migration of hMSC grown in hypoxia for the entire culturing period.
We tested migration toward the factors from three major groups - growth factors, chemokines and inflammatory cytokines - since hMSCs have been shown to express an array of functional receptors for those molecules [35–38]. We found that all 17 cytokines tested in this study mediated in vitro cell migration of hMSC, with the least activity among chemokines and the most among growth factors, which is in agreement with other studies [35–37].
In addition, we noticed that hypoxic cells are more responsive to cytokines often present in a wound milieu (Figure 2). The most important among them are the EGF family, the fibroblast growth factor family, VEGF-121, PDGF-AB, the IL family and TNF-α . The enhanced migration of hMSCs toward the wound healing cytokines can potentially accelerate the formation of new tissue and ultimately wound closure.
In addition, we found a significantly better migration of hypoxic cells toward such factors as HGF, SDF-1α, the IL family cytokines and TNF-α. It is tempting to speculate that this feature may lead to better in vivo homing of long term hypoxia-cultured hMSCs to the sites of infarct and stroke since those factors are abundant at the sites of ischemic injury [39–43].
It is noteworthy that migration of normoxic hMSC transferred for the assay into a hypoxic environment was still lower than in hypoxic cells, but higher than in normoxic cells assessed in normoxia (data not shown) confirming the known stimulating effect of acute hypoxia on the migration of MSC. It has been shown that hypoxic pre-conditioning increases MSC migration  due, at least in part, to up-regulated expression of the chemokine receptors CX3CR1 and CXCR4 , as well as the c-Met receptor for HGF . It is possible that the increased migration of cells permanently cultured in hypoxia can be explained by up-regulation of a series of receptors. However, we believe that the major reason for the mentioned phenomenon is the undifferentiated status of hypoxic MSC.
The latter notion is supported by the analysis of CFU-F efficiency, which demonstrated that hypoxic hMSC have increased CFU-F, compared to similar cells grown under normoxic conditions (Figure 1). Normoxic cells assayed for CFU-F in hypoxia still produced a significantly lower number of colonies compared to hypoxic cells ruling out the effect of assay environment. Rather, the higher number of CFU-F in hypoxic cells may be due to the accumulation of immature stem cell progenitors as previously proposed .
Perhaps the most significant finding of this study is the enhanced activation of RhoA in hypoxic hMSC. The RhoA signaling cascade is believed to play an essential role in the migration of MSC . The family of RhoGTPases directs a variety of cell responses including cell migration, adhesion, transcription, growth and so on . It controls cytoskeletal activation in many cell types including MSC [27, 28]. We demonstrated that upon activation with CN01 the increase of GTP-bound RhoA over non-activated cells is considerably higher in hypoxic hMSC compared to normoxic cells (3-fold and 1.5-fold, respectively). It remains to be investigated whether this phenomenon is true for other types of stem cells reflecting their undifferentiated status. The precise mechanism of the observed difference in sensitivity is unclear. Recently, it has been shown that HIF-1 and RhoA cross-talk suggesting the role of HIF-1 in hypoxic regulation of MSC migration through the RhoA signaling cascade [19, 31].
HIF-1 is one of the key regulators of oxygen homeostasis . HIF-1 is a heterodimeric protein that consists of two subunits, HIF-1α and HIF-1β. While HIF-1β is constitutively expressed, the expression of HIF-1α is lower in normoxic conditions (O2 > 6%) due to its hydroxylation and degradation. In hypoxia (O2 < 6%) both subunits, HIF-1α and β, survive degradation and form an active HIF-1 heterodimer, which binds to the HRE in the cell nucleus . It has been shown that artificial stabilization of HIF-1 in normoxia by desferrioxamine (DFO), as well as pre-conditioning in 1% hypoxia result in attenuation of RhoA activation [19, 31] suggesting that activated HIF-1 is a potential upstream regulator of RhoA. We found that both activated HIF-1 and GTP-bound RhoA were elevated in long term hypoxic cells, reflecting a strong link between HIF-1 and activation of RhoA in hypoxic hMSC.
It should be noted that the RhoA activation pattern observed by Raheja et al.  for hMSC pre-conditioned in 1% hypoxia or treated with DFO (state close to anoxia) is different compared to the data presented in this paper. It can be due to the fact that hypoxia pre-conditioning, which reflects the acute cellular response to hypoxia  rather than cell differentiation status, is substantively different from permanent cell cultivation in the low O2 environment. Also, 0%/1% and 5% oxygen concentrations may potentially have different effects on MSCs physiology . Therefore, we believe it is difficult to directly compare our data to the results reported by Raheja et al. .