# Extracellular Matrix Tunes the Regenerative Potential of Fetal Stem Cells
## 3 Results
### Osteogenic Capacity of Fetal Stem Cells after Growth on dECMs  
To characterize the osteogenic potential of fetal MSCs following expansion on dECMs, P5 fSDSCs and fNPCs, grown on SECM and NECM for one passage, were incubated toward osteogenesis for 21 days. The qPCR data (Figure 6A) showed that after osteogenic induction, the fNPCs responded more significantly than the corresponding fSDSCs and exhibited a higher level of expression of _BGLAP_ and _ALPL_. Compared to the Plastic group, dECM-expanded fNPCs exhibited less _BGLAP_ expression, whereas the dECM-expanded fSDSCs displayed higher _ALPL_ expression. These mRNA data were in line with the  
Figure 4: Chondrogenic staining evaluation of fetal MSCs following expansion on dECMs. fNPCs (N) and fSDSCs (S), following expansion “on” dECMs deposited by fNPCs (NE), fSDSCs (SE), or tissue culture plastic (PL), were incubated in a pellet culture system with chondrogenic medium for 21 days. Alcian blue staining (Ab) was used to detect sulfated GAGs and immunohistochemistry staining (IHC) was used to detect type I collagen (COL1), type II collagen (COL2), and type X collagen (COLX). Scale bar: 400 \(\mu\)m.  
protein expression of ALPL, determined by Western blot analysis (Figure 6B), and calcium deposition, determined by Alizarin Red S staining (Figure 6C). Figure 5: Adipogenic evaluation of dECM-expanded fetal MSCs. Following expansion “on” dECMs deposited by fNPCs (NE), fSDSCs (S), or tissue culture plastic (PL), the fNPCs (N) and fSDSCs (S) were incubated in an adipogenic medium for 21 days. (**A**) TaqMan(r) real-time qPCR was used to evaluate the adipogenic marker genes _LPL_, _FABP4_, and _CEBPA_ in day 21 samples. _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. * indicates a statistically significant difference from the corresponding PL group of cell samples (_p_ < 0.05). (**B**) Western blot analysis was used to evaluate the adipogenic markers PLIN2 and FABP4 of day 21 samples. GAPDH serves as the internal control. (**C**) Oil Red O staining was used to detect the lipid droplets of adipogenically induced cells in T25 flasks. Scale bar: 100 \(\upmu\)m.  
Major Matrix Protein Expression in Fetal Stem Cells, Their Matrix, and Three-Lineage Differentiated Cells  
Immunofluorescence staining analysis showed that the NECM and SECM were positively stained for FN1, PLC, NID1, NID2, LAMA, and COL4. The dECMs from both cells exhibited a comparable density of staining in NID1, NID2, and COL4, except that PLC was intensely stained in SECM and FN1 and LAMA were intensely stained in NECM (Figure 7A). The intensity of dECM staining was in line with the qPCR data of expanded fetal stem cells, in which Plastic-expanded fNPCs expressed more _FN1_ and _LAMA5_ and Plastic-expanded fSDSCs expressed more _HSPG2_ (Figure 7B). We found that after chondogenic induction, _FN1_ and _HSPG2_ were upregulated and _NID1_, _NID2_, _LAMA5_, and _LAMB1_ were downregulated in the fNPCs, while _FN1_ was upregulated and _HSPG2_, _NID1_, _NID2_, and _LAMA5_ were increased in the fNPCs, while _FN1_ was upregulated and _HSPG2_, _NID1_, _NID2_, and _LAMA5_ were increased in the fNPCs, while _FN1_ was suppressed in the fNPCs, while _FN1_ was suppressed in the fNPCs. Figure 6: Osteogenic evaluation of dECM-expanded fetal MSCs. Following expansion “on” dECMs deposited by fNPCs (NE), fSDSCs (SE), or tissue culture plastic (PL), the fNPCs (N) and fSDSCs (S) were incubated in an osteogenic medium for 21 days. (**A**) TaqMan® real-time qPCR was used to evaluate the osteogenic marker genes _BGLAP_ and _ALPL_ of day 21 samples. _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 INPC group.