# Hypoxia Modulates Regenerative Potential of Fetal Stem Cells
## 3 Results
### Effects of Hypoxia on Adipogenic and Osteogenic Differentiation of Fetal Cells  
To determine the direct effect of hypoxia on adipogenic differentiation, after ex vivo expansion in normoxia, fetal cells were cultured in T25 flasks in either normoxia (NN) or hypoxia (NL) for 21-day adipogenic induction. We found that hypoxia dramatically increased expression levels of adipogenic marker genes including _LPL_, _FABP4_, and _CEBPA  
Figure 6: Evaluation of the effect of hypoxia pretreatment on osteogenic potential of fetal MSCs. Fetal NPCs and SDSCs after normoxia (N) or hypoxia treatment (L) were incubated for 21 days in osteogenic induction medium. Osteogenic marker genes _BGLAP_, _SPP1_, _RUNX2_, and _SP7_ 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 (\(p<0.05\)). Alizarin Red S (ARS) staining was used to detect bone nodules (**C**). in both NPCs and SDSCs (Figure 8A). NPCs were less responsive to hypoxia compared to SDSCs (Figure 8A), which was supported by western blot results, in which FABP4 was highly expressed in adipogenically induced SDSCs (Figure 8B). ORO staining data indicated that more lipid droplets were found in adipogenically induced SDSCs compared to NPCs and fetal cells treated with hypoxia compared to normoxia (Figure 8C), which further confirmed the mRNA data (Figure 8A). To determine the direct effect of hypoxia on osteogenic differentiation, after ex vivo expansion in normoxia, fetal cells were cultured in T25 flasks in either normoxia (NN) or hypoxia (NL) for 21-day osteogenic induction. We found that hypoxia decreased expression levels of osteogenic marker genes including _BGLAP_ and _SPP1_ in both NPCs and SDSCs; intriguingly, no significant difference was found in _RUNX2_ and _SPP_ expression in both cell types (Figure 9A). ARS staining also confirmed that hypoxia decreased bone nodules in both cell types (Figure 9B). Figure 7: Evaluation of the effect of hypoxia on chondrogenic differentiation of fetal MSCs. RT-qPCR was used to assess the expression of chondrogenic marker genes (_SOX39_, _ACAN_, _COL2A1_, and _PRG4_) (**A**) and chondrogenic related genes (_FOXF1_, _FBLN1_, _COL1A1_, and _COL10A1_) (**B**) in days 0, 10, and 21 pellets, 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 group within the same cell type (\(p<0.05\)). Histology (**C**) using Alican blue staining (Ab) for sulfated GAG and immunohistochemical staining (IHC) for type II collagen (Col2) and type I collagen (Col1) along with a gross photo to indicate the size of pellet (mm) in day 21 chondrogenically induced pellets. Figure 8: Evaluation of the effect of hypoxia on adipogenic differentiation of fetal MSCs. Normoxia expanded NPCs and SDSCs were incubated in adipogenic medium for 21 days in either normoxia (NN) or hypoxia (NL). RT-qPCR was used to evaluate the expression of adipogenic marker genes (_LPL_, _CEBPA_, _FABP_, and _PPARG_) (**A**), 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 group within the same cell type (_p_ < 0.05). Western blot was used to assess the expression of the FABP4 protein (**B**). GAPDH served as an internal control. Oil Red O (ORO) staining was used to stain lipid droplets (**C**). Figure 9: Evaluation of the effect of hypoxia on osteogenic differentiation of fetal MSCs. Normoxia expanded NPCs and SDSCs were incubated in osteogenic medium for 21 days in either normoxia (NN) or hypoxia (NL).