# Hypoxia Modulates Regenerative Potential of Fetal Stem Cells
## 3 Results
### Effects of Hypoxia on Chondrogenic Differentiation of Fetal Cells  
To determine the direct effect of hypoxia on chondrogenic differentiation, after ex vivo expansion in normoxia, fetal cells were cultured in a pellet system in either normoxia (N-N-N), hypoxia (N-L-L), or partial hypoxia (N-N-L) for 21 days. We found that the N-L-L pellets exhibited the highest expression levels of chondrogenic marker genes including _SOX9_, _ACAN_, _COL2A1_, and _PRG4_ (Figure 7A). Despite comparable expression of _SOX9_, _ACAN_, and _COL2A1_ in day 0 NPC and SDSC pellets, NPC pellets exhibited a greater  
Figure 4: Evaluation of the effect of hypoxia pretreatment on the chondrogenic potential of fetal MSCs using histology and immunohistochemical staining. NPC (**A**) and SDSC (**B**) pellets were cultured in chondrogenic induction medium for 21 days. Hypoxia was used in cell expansion and subsequent induction in different combinations (see Figure 1A). Histology using Alican blue staining (Ab) for sulfated GAG (with fast red as a counterstained) and immunohistochemical staining (IHC) for type II collagen (Col2) and type I collagen (Col1) (with hematoxylin as a counterstainedain) along with a gross photo to indicate the size of the pellet (mm).  
level of expression at both day 10 and day 21 than the SDSC counterparts, particularly for hypoxia treated pellets (Figure 7A). Intriguingly, despite a lower level of _PRG4_ expression in day 0 NPC pellets, 21-day chondrogenic induction dramatically increased its expression in NPC pellets compared to the SDSC counterpart, particularly for hypoxia-treated pellets (Figure 7A).  
Interestingly, we found another two related markers _FOXF1_ and _FBLN1_, previously demonstrated to be primarily in nucleus pulposus and articular cartilage, respectively [22], responded to hypoxia differently from the above chondrogenic gene expression. Despite higher expression of _FOXF1_ in day 0 NPC pellets compared to SDSC pellets, chondrogenic induction dramatically decreased its expression at day 10 followed by an increase at day 21 in NPC pellets while SDSC pellets exhibited a continued decrease for up to 21 days (Figure 7B). We also found that day 0 NPC pellets exhibited comparable expression of _FBLN1_ to SDSC pellets (Figure 7B) despite higher levels in SDSC versus NPC cell samples (Figure 3C). Despite the fact that chondrogenic induction favored _FBLN1_ expression in day 21 NPC pellets, hypoxia decreased expression of _FOXF1_ and _FBLN1_ as well as _COLIA1_ and _COLI0A1_ in both NPC and SDSC pellets under chondrogenic induction (Figure 7B).  
Figure 5: Evaluation of the effect of hypoxia pretreatment on adipogenic potential of fetal MSCs. Fetal NPCs and SDSCs after normoxia (N) or hypoxia treatment (L) were incubated for 21 days in adipogenic induction medium. Adipogenic marker genes _LPL_, _FABP4_, _PPARG_, and _CEBPA_ were assessed using RT-qPCR before (**A**: cell sample) and after induction (**B**: day 21 samples), normalized against _GAPDH_ levels as an internal control. Data (\(n=4\)) are represented in bar charts. Different letters indicate a statistically significant difference compared to the groups within the same gene type (\(p<0.05\)). Western blot was used to detect FABP4 expression (**C**) in fetal MSCs after adipogenic induction. GAPDH was used as an internal control. Oil Red O (ORO) staining was used to detect lipid droplets (**D**).  
After 21-day chondrogenic induction, the L-L-L pellets showed the largest size and the most intensity of Alcian blue staining for sulfated GAGs and immunohistochemical staining for type II collagen whereas the N-N-N pellets displayed the most intensity of immunohistochemical staining for type I collagen (Figure 7C), which is in line with the RT-qPCR results (Figure 7A,B). We also found that SDSC pellets showed less intensity of both sulfated GAGs and type II collagen staining (Figure 7C).