# Extracellular Matrix Tunes the Regenerative Potential of Fetal Stem Cells
## 4 Discussion  
Fetal MSCs are a promising approach to stem-cell therapy in regenerative orthopedic medicine [25]. An ex vivo environment, such as hypoxia, has been used to modulate the  
Figure 7: Major matrix protein expression in dECMs and differentiated fetal MSCs. (**A**) Immunofluorescent resonance staining was used to detect the expression of FN1, PLC, NID1, NID2, LAMA, and COL4 in dECMs deposited by fNPCs or fSDSCs. Scale bar: 100 \(\upmu\)m. (**B**) TaqMan® real-time qPCR was used to evaluate the mRNA levels of _FN1_, _HSPG2_, _NID1_, _NID2_, _LAMA5_, and _LAMB1_ in expanded fetal MSCs and in chondrogenically, adipogenically, and osteogenesisly induced cells. _GAPDH_ served as the internal control. Data are shown as a bar chart. * indicates a statistically significant difference from the corresponding PL group (\(p<0.05\)). * indicates a statistically significant difference from the corresponding fNPC group (\(p<0.05\)). * indicates a statistically significant difference from the corresponding PL expanded cell samples (\(p<0.05\)). - regenerative potential of fetal stem cells [23]. Knowing that a matrix microenvironment can influence the therapeutic potential of adult stem cells [11; 26], in this study, we characterized two fetal cell types for their response to expansion on fetal dECM and the subsequent differentiation capacity. Compared to fNPCs, the fSDSCs showed a higher overall expression of MSC surface markers, indicating greater cell proliferation capacity. Interestingly, fSDSCs grown on SECM exhibited an increased expression of CD73, SSEA4, and relative EdU incorporation but a decreased expression of CD90, CD105, and CD146. This finding is in line with previous reports, in which aSDSCs or fSDSCs grown on SECMs and deposited by either aSDSCs or fSDSCs displayed a decrease in expression of CD90 and CD105 but an increase in SSEA4 [19; 27]. An increase in the expression of SSEA4 and relative EdU incorporation indicates that dECM expansion promoted MSC proliferation. The International Society for Cellular Therapy's 2006 conference defined CD73, CD90, and CD105 as surface markers for human MSCs [28]. Given this definition, it is challenging to interpret the similar trend in the expression of MSC surface markers following dECM expansion with the dissimilar differentiation capacities shown in this study and others [29]. We also found that fetal cells responded differently to matrix rejuvenation in stemness gene expression. While fSDSCs are considered closer to MSCs and, therefore, more pluripotent than fNPCs, after dECM rejuvenation, interestingly, the fNPCs displayed higher levels of pluripotency gene expression than the fSDSCs. Following expansion on Plastic flasks, different from aSDSCs losing their chondogenic capacity during passaging from P3 to P5 [30], the fSDSCs regained their chondogenic potential from P2 to P9 [27]. dECM rejuvenation is of benefit to adult MSCs grown on fetal dECM [19] and fetal MSCs grown on adult dECM [27] but not to adult MSCs expanded on old dECM [20] and fetal MSCs expanded on fetal dECM [27]. In this study, fSDSCs grown on dECMs deposited by fSDSCs and fNPCs yielded 21-day pellets with a decreased expression of _SOX9_ and _ACAN_ and increased expression of _COL2A1_, with no significant difference in _PRG4_ and _FBLN1_. In contrast, fNPCs grown on dECMs and deposited by fSDSCs and fNPCs yielded 21-day pellets with an increased expression of _ACAN_, _COL2A1_, and _PRG4_, and a decreased expression of _COL1A1_ and _COL10A1_. This finding indicates that, despite the fact that both are fetal cells, fNPCs (a progenitor cell for NP tissue) responded more positively to the rejuvenation of dECMs deposited by fSDSCs and fNPCs than fSDSCs. Interestingly, dECM-rejuvenated fNPCs exhibited a less significant response to both osteogenic and adipogenic induction compared to the Plastic group, which may be associated with the inherent nature of NPCs [31]. Compared to fNPCs, fSDSCs are closer to fetal MSCs.