# Hypoxia Modulates Regenerative Potential of Fetal Stem Cells
## 3 Results
### Effects of Hypoxia Pretreatment on Chondrogenic Capacity of Fetal Cells  
To determine the influence of hypoxia on chondrogenic gene expression of both fetal cells, major chondrogenic marker genes including _SOX9_, _ACAN_, _COL2A1_, and _PRG4_ were evaluated using RT-qPCR. The data showed that hypoxia decreased expression of _ACAN_ and _PRG4_ in SDSCs despite there being no significant difference in NPCs (Figure 3A). After 21-day chondrogenic induction in a pellet culture system, we found that hypoxia pretreatment significantly increased chondrogenic marker gene (_ACAN_, _COL2A1_, and _PRG4_) expression in NPCs compared to the normoxia group (L-N-N versus N-N-N) despite nonsignificant expression of _SOX9_ (Figure 3B). Interestingly, exposure to low oxygen in the early phase of chondrogenic induction (D0-D10) significantly increased _SOX9_, _ACAN_, and _PRG4_ expression compared to the hypoxia pretreatment group (N-L-N versus L-N-N) (Figure 3B). We also found that hypoxia treatment throughout both the expansion and differentiation phases (L-L-L) yielded NPCs with the highest expression of _SOX9_, _ACAN_, and _COL2A1_ (Figure 3B). SDSCs responded to hypoxia differently compared to NPCs (Figure 3B). We found that hypoxia pretreatment increased _SOX9_ and _COL2A1_ expression but decreased _ACAN_ and _PRG4_ levels compared to normoxia treatment. Exposure to low oxygen in the early phase of chondrogenic induction (D0-D10) promoted _COL2A1_ and _PRG4_ compared to the normoxia group and hypoxia preconditioned groups (N-L-N versus N-N-N/L-N-N), which may have significance clinically given the fact that _PRG4_ encodes lubricin, a secreted protein that helps in joint lubrication and movement. Hypoxia treatment throughout both the expansion and differentiation phases (L-L-L) yielded SDSCs with the highest expression of _COL2A1_. Overall, SDSCs exhibited a weaker response to chondrogenic induction than NPCs in both normoxic and hypoxic conditions. We also assessed other chondrogenesis-associated gene expressions in response to hypoxia pretreatment, including _FBLN1_ (articular cartilage marker gene), _FOXF1_ (NPC marker gene), _CDH2_ (mesenchymal condensation marker gene), and _FN1_ (cell condensation associated gene) (Figure 3C). We found that NPCs exhibited higher expression of _FOXF1_ while SDSCs displayed higher expression of _FBLN1_, which is in line with previous reports that _FOXF1_ was highly expressed in human NP while _FBLN1_ was advantageously expressed in human articular cartilage [22]; SDSCs are a tissue-specific stem cell for chondrogenesis [14]. NPCs also exhibited significant expression of _CDH2_ and _FN1_ compared to SDSCs, indicating the greater chondrogenic potential of NPCs [23; 24]. Interestingly, hypoxia pretreatment decreased most of these condensation gene expressions, particularly for SDSCs. After 21-day chondrogenic induction, we found that hypoxia pretreatment of NPCs exhibited the highest expression of _FOXF1_ and _FBLN1_ as well as _COL1A1_ and _COL10A1_ while SDSCs displayed the highest expression of _FBLN1_ as well as _COL10A1_ (Figure 3D). Interestingly, hypoxia treatment throughout both the expansion and differentiation phases (L-L-L) significantly decreased early chondrogenic marker _COL1A1_ and hypertrophic marker _COL10A1_ expression in both NPCs and SDSCs. Histologically, NPC pellets were more dense in Alcian blue staining (Ab) for sulfated GAG and immunohistochemical staining (IHC) for type II collagen compared to SDSC pellets with a larger size, particularly for the hypoxia treatment group (L-L-L) (Figure 4A versus Figure 4B), which is in line with the above RT-qPCR data. Intriguingly, the hypoxia pretreatment group yielded NPC pellets with the weakest staining of sulfated GAG and type II collagen (Figure 4A).