# Extracellular Matrix Tunes the Regenerative Potential of Fetal Stem Cells
## 2 Materials & Methods
### Induction and Assessment of Adipogenesis, Osteogenesis, and Chondrogenesis  
For chondrogenic induction, 0.3 \(\times\) 10\({}^{6}\) expanded cells from each group were centrifuged at 1200 rpm for 7 min in a 15-mL polypropylene tube to make a pellet. After 24 h of incubation in complete medium, D0 pellets were collected. The remaining pellets were cultured in a serum-free chondrogenic induction medium consisting of high-glucose Dulbecco's Modified Eagle's medium (DMEM), 40 \(\mathrm{\SIUnitSymbolMicro g}\)/mL proline (Millipore Sigma), 100 \(\mathrm{\SIUnitSymbolMicro m}\) dexamethasone (Millipore Sigma), 1\(\times\) PSF, 0.1 mM ascorbic acid-2-phosphate (Wako Chemicals), and 1 \(\times\) ITS\({}^{\mathrm{TM}}\) Premix (BD Biosciences), with the supplementation of 10 \(\mathrm{\SIUnitSymbolMicro g}\)/mL of transforming growth factor beta 3 (TGF-63; PeproTech Inc., Rocky Hill, NJ, USA) in a 37 \({}^{\circ}\)C, 5\(\mathrm{\SIUnitSymbolMicro O}\)\({}_{2}\), 5\(\mathrm{\SIUnitSymbolMicro O}\)\({}_{2}\) humidified incubator for 21 days. Chondrogenic differentiation was evaluated using histology and immunohistochemistry (IHC) analyses, as well as the qPCR targeting of chondrogenic-related genes (Table 2). For histology, the representative pellets (n = 3) were fixed in 4% paraformaldehyde at 4 \({}^{\circ}\)C overnight, followed by dehydration in a gradient ethanol series, clearing with xylene, and embedding in paraffin blocks. For histological staining, 5-\(\mathrm{\SIUnitSymbolMicro m}\)-thick sections were stained with Alican blue (Thermo Fisher Scientific) for sulfated glycosaminoglycan (GAG) and counterstainedated with fast red for nuclei. For IHC analysis, the consecutive sections were probed with primary antibodies against type I collagen (GeneTex, Irvine, CA, \begin{table}
\begin{tabular}{c c c} \hline
**Gene Name** & **Full Name** & **TaqMan® Assay ID** \\ \hline \multicolumn{3}{c}{_Chondrogenesis-related genes_} \\ \hline _SOX9_ & SRY-Box 9 & Hs00165814\_m1 \\ _ACAN_ & Aggrecan & Hs00153936\_m1 \\ _Col2 AI_1 & Type II collagen & Hs00156568\_m1 \\ _PRG4_ & Proteoglycan 4 & Hs0081633\_m1 \\ _FBLN1_ & Fibulin 1 & Hs00972609\_m1 \\ _FOXF1_ & Forkhead box F1 & Hs00230962\_m1 \\ \hline \multicolumn{3}{c}{_Housekeeping internal gene_} \\ \hline _GAPDH_ & Glyceraldehyde-3-phosphate dehydrogenase & Hs02758991\_g1 \\ \hline \end{tabular}
\end{table} 
Table 2: _Cont._ \begin{table}
\begin{tabular}{c c c c} \hline
**Antibody** & **Catalog No.** & **Company** & **Species** & **Working Conc.** \\ \hline Type II collagen & II6B3-c & Developmental Studies Hybridoma & Mouse & 3 \(\mathrm{\SIUnitSymbolMicro g}\)/mL \\ Type IV collagen & M3F7 & Bank (DSHB), Iowa City, IA, USA & Mouse & 3 \(\mathrm{\SIUnitSymbolMicro g}\)/mL \\ Fibronectin & HFN7.1 & Mouse & 3 \(\mathrm{\SIUnitSymbolMicro g}\)/mL \\ Laminin & PAI-16730 & Invitrogen, Waltham, MA, USA & Rabbit & 20 \(\mathrm{\SIUnitSymbolMicro g}\)/mL \\ Col1A1 (3G3) & sc-293182 & & Mouse & 1 \(\mathrm{\SIUnitSymbolMicro g}\)/mL \\ Perlecan (A7L6) & sc-33707 & Santa Cruz Biotechnology, Inc., & Rat & 4 \(\mathrm{\SIUnitSymbolMicro g}\)/mL \\ Nidogen 1 (C-7) & sc-133175 & Dallas, TX, USA & Mouse & 2 \(\mathrm{\SIUnitSymbolMicro g}\)/mL \\ Nidogen-2 (F-2) & sc-377424 & & Mouse & 2 \(\mathrm{\SIUnitSymbolMicro g}\)/mL \\ \hline \end{tabular}
\end{table} 
Table 3: Primary antibodies used in the immunofluorescence staining of dECMs. USA), type II collagen (Developmental Studies Hybridoma Bank, DSHB, Iowa City, IA, USA), and type X collagen (GeneTex), followed by the secondary antibody of biotinylated horse anti-mouse/rabbit IgG (H + L) (Vector, Burlingame, CA, USA). Immunoactivity was detected using Vectastain ABC reagent (Vector), with 3, 3'-diaminobenzzidine (DAB, Vector) as a substrate. Counterstaining was performed with hematoxylin (Vector).