# Hypoxia Modulates Regenerative Potential of Fetal Stem Cells
## 4 Discussion  
Compared to adult stem cells, which have been well-studied in regard to the hypoxic influence on both proliferation and differentiation, few reports have focused on fetal stem cells. The objective of this study was to characterize the regenerative potential of fetal cells under hypoxia. Due to the importance of hypoxia in cartilage development and regeneration, two fetal cell sources were chosen for chondrogenic evaluation based on their associations with cartilage. Given their tremendous regenerative potential, both fetal cell types were also evaluated for their adipogenic and osteogenic potential/differentiation under hypoxia. We found that hypoxia during both cell expansion and differentiation phases increased chondrogenic potential and differentiation, respectively. Interestingly, while adipogenic capacity was increased upon both hypoxic expansion and differentiation, osteogenic capacity was basically unchanged following hypoxic expansion and decreased upon hypoxic differentiation. We found hypoxia increased cell proliferation and expression of MSC surface markers, suggesting that hypoxia helps to maintain fetal cell plasticity and suppress random differentiation, similar to previous studies in adult MSCs in which hypoxia preconditioning prevented cell senescence while enhancing proliferation, differentiation, and migration [9; 25; 26]. Our data suggested that hypoxic pretreatment not only significantly increased chondrogenic marker gene expression but also had the lowest levels of types I and X collagen expression in both fetal MSCs, which indicates that hypoxia preconditioning facilitates cartilage phenotypic stability and maturation. This finding aligns with a previous report that hypoxia has an anabolic effect on adult MSC chondrogenesis that upregulates chondrogenic genes and downregulates hypertrophic genes [27]. Potential mechanisms associated with hypoxia signals were demonstrated through hypoxia transcription factors (HIFs), particularly HIF1\(\alpha\) and HIF2\(\alpha\), and the PI3K/Akt/FoxO pathways [16]. These pathways help to mediate cellular responses to oxygen levels and regulate inflammatory cytokines. For instance, when evaluating hypoxic prechondrogenic cells in mice, HIF1\(\alpha\) activity has been linked to a two-fold increase of _SOX9_ expression in hypoxic conditions when compared to normoxic conditions [28]. In human articular chondrocytes, _HIF2\(\alpha\)_ knockout has been shown to decrease chondrogenic gene expression [29]. We found that although both cell types demonstrated increased chondrogenic potential in response to hypoxic pretreatment, the two fetal cell types displayed different sensitivities to chondrogenic induction, with NPCs displaying the more intense response. When comparing fetal NPC and SDSC responses to low oxygen, NPCs appear to be more sensitive to low oxygen, as they exhibited increasing gene expression going from day 10 to day 21 pellets. In contrast, SDSC day 10 pellets showed high chondrogenic differentiation marker expression, but then demonstrated a slight downward trend in going from day 10 to day 21 pellets. Our finding is consistent with previous reports comparing human adult NPCs and other sources of MSCs; for instance, adult NPCs have previously been compared to adult bone marrow-derived MSCs (BMSCs) in regard to their chondrogenic, adipogenic, and osteogenic differentiation potentials, where they demonstrated similarly higher chondrogenic differentiation capacity [30]. Interestingly, we found that chondrogenic induction with hypoxia treatment, either in the early stage (N-L-N) or in the whole differentiation stage (N-L-L), yielded 21-day pellets with the highest expression level of _PRG4_ in both fetal cell groups. Given that lubricin is a secreted protein that helps in joint lubrication and movement, 21-day NPC pellets with a higher expression level of _PRG4_ compared to SDSC counterparts suggest that fetal NPCs might be a better cell source for articular cartilage regeneration.