To gain a better understanding of the molecular mechanisms that control cellular responses of progenitor cells to hypoxia, we compared changes in global gene expression in UCB CD133 + cells and BMMC cultured in normoxic or hypoxic conditions for 24 hours. Differential gene expression regulated by hypoxia was determined by comparing pairwise normoxic and hypoxic samples for each cell population as described in Materials and Methods. The cut-off limits used by others for a significant fold change vary from threefold  to 1.85-fold  to 1.5-fold . When we used a threshold of 1.5-fold, a total of 214 and 92 genes appeared to be differentially regulated by hypoxia at 24 hours in UCB CD1133+ cells and BMMCs, respectively (data not shown). We decided to use a threshold of 1.75-fold change because this cut-off included many of the genes known to be regulated by hypoxia (e.g., VEGFA, PGK1, and ENO2.). On the basis of this criterion, we identified a total of 183 genes differentially regulated by hypoxia at 24 hours in UCB CD133+ cells. Approximately 161 genes of the total 183 were upregulated (> 1.75-fold), whereas 22 genes were downregulated. Annotations were performed using GO, and the candidate genes were classified into categories according to biological processes. Figure 3A shows that 26.8% of the genes differentially regulated by hypoxia in UCB CD133+ progenitor cells have not been assigned to any biological process (unknown), and almost half of the genes (48%) were involved in one of the following categories: metabolism (13.1%), cell proliferation/survival (10.4%), transcription (9.3%), signal transduction (7.7%), and transport (6.6%). In BMMC, 45 genes were differentially regulated by hypoxia at 24 hours. Thirty-three  of these genes were upregulated by more than 1.75-fold, and 12 genes were downregulated. After we categorized genes using GO (Fig. 3B), approximately 26.7% of the genes differentially regulated by hypoxia in BMMC had unknown associated biological processes, and more than half (51.1%) could be assigned to one of the following categories: signal transduction (15.5%), cell proliferation/survival (8.9%), metabolism (8.9%), transcription (8.9%), and nucleic acid metabolism (8.9%). When hypoxia-responsive genes found in UCB CD133+ cells and BMMC were compared, only nine such genes were shared between the two progenitor cell populations. These included ADM, AK3L1/L2, BNIP3, ENO2, PGK1, SLC16A3, SOS2, TPI, and VEGFA (Table 1). Seven of the nine genes are known to be regulated by hypoxia in other cell types, such as endothelial or cancer cells [42, , , , , , , , –51]. The exceptions are AK3L1/L2, which is involved in metabolism, and SOS2, which is associated with signal transduction in Drosophila . Three of the eight genes shared between UCB CD133+ cells and BMMC are involved in metabolism (ENO2, TPI, and PGK1), two are associated with cell proliferation and survival (VEGFA and BNIP3), and the rest are involved in transport (SLC16A3) and signal transduction (ADM). Taken together, these results show that only a small proportion of genes regulated by hypoxia are common to UCB CD133+ cells and BMMC, suggesting that their molecular response to low oxygen levels is largely cell type-specific.
Interestingly, genes involved in cell proliferation and survival represented one of the largest categories of genes regulated by hypoxia in both UCB CD133+ cells and BMMC. In BMMC, these included BNIP3, DUSP1, FGF2, and VEGFA. BNIP3 is a proapoptotic factor known to be regulated by hypoxia in cancer cells . DUSP1 encodes a dual specific phosphatase that protects against overactivation of HIF-1α , and possibly against HIF-1α-mediated cell cycle arrest [54, 55]. FGF2 and VEGFA are growth factors with potent proangiogenic and mitogenic characteristics that may exert paracrine and autocrine effects. Recently, it has been suggested that VEGFA has an autocrine mitogenic role in human bone marrow-derived mesenchymal stem cells . In contrast to our results with BMMC, the overall proliferation of UCB CD133+ cells remained unaltered after 24-hour exposure to hypoxia, although we noted an increase in clonogenic potential. Genes differentially regulated by hypoxia associated with cell proliferation/survival in UCB CD133+ cells included, among others CCNG2, a negative regulator of cell cycle progression  and NEDD9, which is expressed in G1 phase arrest , suggesting that there might be a transient block in cell cycle progression during the exposure to hypoxia.
The osteogenic and adipogenic differentiation potential of BMMC exposed to hypoxia for 24 hours was unaltered, whereas the chondrogenic potential was increased. Consistent with these results, the mRNA levels of the adipogenic factor PPAR-α and osteogenic factor RUNX2 were unchanged in BMMC exposed to hypoxia. By contrast, the expression of SOX9, a key transcription factor responsible for chondrocyte differentiation, was increased by 1.54-fold (Fig. 1G). A similar increase in SOX9 expression was observed in the microarray analysis (1.66-fold). Recently, it has been reported that hypoxia induces Sox9 gene expression in murine mesenchymal cells .