# Extracellular Matrix Tunes the Regenerative Potential of Fetal Stem Cells
## 2 Materials & Methods
### Evaluation of Proliferation, Surface Markers, and Stemness Genes of Expanded fSDSCs and fNPCs  
P5 fSDSCs and P5 fNPCs were expanded on Plastic and dECMs deposited by fSDSCs and fNPCs, respectively, for one passage, subsequently followed by the analyses below.  
The expanded cells were assessed for cell proliferation using a nucleoside analog Click-iT EdU Alexa Flour(tm) 647 flow cytometry assay kit (Invitrogen, Eugene, OR, USA). After the expanded cells reached 45% confluence, EdU was added to the cell culture medium at a final concentration of 10 uM. After 20 h of incubation, the harvested cells (3 \(\times\) 10\({}^{5}\) in each group) were incubated with Click-iT(tm) reaction cocktail (1\(\times\)) in the dark for 30 min. Fluorescence was detected by a BD FACSCalibur(tm) flow cytometer (BD Biosciences, San Jose, CA, USA). The FCS Express 7 Research Edition 7.18.0025 (De Novo Software, Los Angeles, CA, USA) was used for data analysis.  
Expanded cells (4 \(\times\) 10\({}^{5}\) in each group) were incubated in cold PBS containing 0.1% ChromPure Human IgG for 30 min. Then, the cells were incubated in the dark with primary antibodies at 4 \({}^{\circ}\)C for 30 min. The primary antibodies used are listed in Table 1.  
Fluorescence was analyzed by a BD FACSCalibur(tm) flow cytometer (BD Biosciences). Data analysis was performed by the FCS Express 7 Research Edition (De Novo software).  
Total RNAs from the samples (n = 3), extracted using Trizol(r) (Millipore Sigma), were quantified, and about 2 ug of total RNA were used for reverse transcription with a high-capacity cDNA reverse transcription kit at 37 \({}^{\circ}\)C for 120 min, as recommended by the manufacturer (Thermo Fisher Scientific). A TaqMan(r) real-time quantitative polymerase chain reaction (qPCR) was used to evaluate the expression of stemness-related genes (_MYC_, _KLF4_, _BMI1_, _POU5F1_, _NES_, _NOV_, _NANOG_, and _SOX2_), adipogenic-related genes (_LPL_, _FABP4_, and _CEBPA_), osteogenic-related genes (_BGLAP_, _ALPL_, and _COLIA1_), and chondogenic marker-related genes (_SOX9_, _ACAN_, _Col2A1_, _PRG4_, _FBLN1_, and _FOXF1_). _GAPDH_ was utilized as the endogenous control gene. The TaqMan(r) Assay IDs of the primers are listed in Table 2. Each experiment was repeated three times using the Applied Biosystems(tm) 7500 fast real-time PCR system (Applied Biosystems, Waltham, MA, USA). Relative transcript levels were calculated as \(\chi=2^{-\Delta\text{CI}}\), in which \(\Delta\Delta\text{CI}=\Delta\text{E}-\Delta\text{C}\), \(\Delta\text{E}=\text{C}\text{te}_{\text{xp}}-\text{C}\text{t}_{\text{GAPDH}}\), and \(\Delta\text{C}=\text{C}\text{t}_{\text{CLI}}-\text{C}\text{t}_{\text{GAPDH}}\).  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Gene Name** & **Full Name** & **TaqMan® Assay ID** \\ \hline \multicolumn{4}{c}{_Stemness-related genes_} \\ \hline _MYC_ & MYC proto-oncogene & Hs00153408\_m1 \\ _KLF4_ & Kruppel-like factor 4 & Hs00358836\_m1 \\ _BMI1_ & B lymphoma Mo-MLV insertion region 1 homolog & Hs00180411\_m1 \\ _POU5F1_ & POU class 5 homeobox 1 & Hs04260367\_gH \\ _NES_ & Nestin & Hs04187831\_g1 \\ _NOV_ & Nephroblastoma overexpressed & Hs00159631\_m1 \\ _NANOG_ & Nanog homeobox & Hs02387400\_g1 \\ _SOX2_ & SRY-box2 & Hs01053049\_s1 \\ \hline \multicolumn{4}{c}{_Adiogenesis-related genes_} \\ \hline _LPL_ & Lipoprotein lipase & Hs00173425\_m1 \\ _FABP4_ & Fatty acid-binding protein 4 & Hs01086177\_m1 \\ _CEBPA_ & CCAAT/enhancer-binding protein alpha & Hs00269972\_s1 \\ \hline \multicolumn{4}{c}{_Osteogenesis-related genes_} \\ \hline _BGLAP_ & Bone gamma-carboxyglutamate protein & Hs01587814\_g1 \\ _ALPL_ & Alkaline phosphatase, liver & Hs01029144\_m1 \\ _COLIA1_ & Type I collagen & Hs00164004\_m1 \\ \hline \hline \end{tabular}
\end{table}
Table 2: TaqMan® assay ID information of the target genes for qPCR.