Day 2 hMSC sheets (n = 3 per group) were homogenized in TRI Reagent (Sigma-Aldrich) for subsequent total RNA extraction and complementary DNA (cDNA) synthesis (iScript Kit, Bio-Rad, Hercules, CA). One hundred nanograms of cDNA was amplified in duplicates in each 40-cycle reaction using a Mastercycler (Eppendorf, Hauppauge, NY) with annealing temperature set at 60°C, SYBR Premix Ex Taq II (Takara Bio Inc., Kusatsu, Shiga, Japan) and custom-designed quantitative reverse transcription polymerase chain reaction (qRT-PCR) primers (Life Technologies, Grand Island, NY; table S1). Transcript levels were normalized to glyceraldehyde-3-phosphate dehydrogenase, and gene expression was calculated as fold change using the comparative CT method (63).