# Hypoxia Modulates Regenerative Potential of Fetal Stem Cells
## 2 Materials and Methods
### Three-Lineage Differentiation  
#### 2.3.1 Chondrogenic Induction and Evaluation  
To induce chondrogenic differentiation, pellet models were formed by centrifuging an aliquot of 0.4 \(\times\) 10\({}^{6}\) expanded cells at 500\(\times\) g for 7 min in a 15-mL polypropylene tube. Pellets were then cultured in a serum-free high-glucose Dulbecco's Modified Eagle's Medium (DMEM) with 100 U/mL penicillin, 100 ug/mL streptomycin, 40 ug/mL proline (Millipore Sigma), 100 nM dexamethasone (Millipore Sigma), 0.1 mM ascorbic acid-2-phosphate (Thermo Fisher Scientific), ITST\({}^{\text{m}}\) Premix (BD Biosciences), and 10 ng/mL transforming growth factor beta 3 (TGF\(\beta\)3; PeproTech, Rocky Hill, NJ, USA). The pellets were collected at days 0, 10, and 21 for histology, immunohistochemistry, and RT-qPCR (see Section 2.2.3 for details).  
For histology, representative pellets (\(n=3\)) were fixed overnight in 4% paraformaldehyde-hyde at 4 \({}^{\circ}\)C, followed by dehydrating through a gradient ethanol series, clearing with xylene, and embedding in paraffin blocks. Then, 5 um sections of the pellets were stained with Alcian blue (Millipore Sigma) (counterstained with fast red) for sulfated glycosaminoglycans (GAGs). For immunohistochemistry, the sections were immunabeled with primary antibodies against type II collagen (catalog number II-II6B3; Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA, USA) and type I collagen (catalog number GTX26308; DSHB), followed by the secondary antibody of biotinylated horse anti-mouse IgG (Vector, Burlingame, CA, USA). Immunoactivity was detected using Vectastain ABC reagent (Vector) with 3,3\({}^{\prime}\)-diaminobenzidine as a substrate.  
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Gene Name** & **Full Name** & **Assay ID** \\ \hline \multicolumn{3}{c}{Stemness related genes} \\ BMI1 & B lymphoma Mo-MLV insertion region 1 homolog & Hs00180411\_m1 \\ KLF4 & Kruppel-like factor 4 & Hs00358836\_m1 \\ MYC & MYC proto-oncogene & Hs00153408\_m1 \\ NANOG & Nanog Homebox & Hs02387400\_g1 \\ NES & Nestin & Hs04187831\_g1 \\ NOV & Nephroblastoma overexpressed & Hs00159631\_m1 \\ POU5F1 & POU class 5 homeobox 1 & Hs04260367\_gH \\ SOX2 & SRY-box 2 & Hs01053049\_s1 \\ Chondrogenesis related genes & \\ _ACAN_ & Aggrecan & Hs00153936\_m1 \\ _CDH2_ & Cadherin 2 & Hs00983056\_m1 \\ _COL2A1_ & Type II collagen & Hs00156568\_m1 \\ _COL1A1_ & Type I collagen & Hs00164004\_m1 \\ _COL10A1_ & Type X collagen & Hs00166657\_m1 \\ _PRG4_ & Proteoglycan 4 & Hs00981633\_m1 \\ _SOX9_ & SRY-Box 9 & Hs00165814\_m1 \\ _CAPB_ & Adipose related genes & \\ _CEBPA_ & CCAAT/enhancer-binding protein alpha & Hs00269972\_s1 \\ _FABP4_ & Fatty acid-binding protein 4 & Hs01086177\_m1 \\ _LPL_ & Lipoprotein lipase & Hs00173425\_m1 \\ _PPARG_ & Peroxisome Proliferator Activated Receptor Gamma & Hs01115513\_m1 \\ \multicolumn{3}{c}{Osteogenesis related genes} \\ _BGLAP_ & Osteocalcin & Hs01587814\_g1 \\ _SPP1_ & Osteopontin & Hs00959010\_m1 \\ _SPP_ & Osterix & Hs01866874\_s1 \\ _RUNX2_ & Runt-related transcription factor 2 & Hs00231692\_m1 \\ \multicolumn{3}{c}{Other related genes} \\ _FBLN1_ & Fibulin 1 & Hs00972609\_m1 \\ _FN1_ & Fibronectin 1 & Hs01549976\_m1 \\ _FOXF1_ & Forkhead Box F1 & Hs00230962\_m1 \\ _GAPDH_ & Glyceraldehyde-3-phosphate dehydrogenase & Hs02758991\_g1 \\ _HSPG2_ & Heparan Sulfate Proteoglycan 2 & Hs01078536\_m1 \\ \hline \hline \end{tabular}
\end{table}
Table 1: Target genes’ Assay ID information for RT-qPCR.