Retroviral and Lentiviral Transduction of hUCB-MSCs

The retroviral plasmid vectors, including pBABE-puro–control, pBABE-puro–GFP–wt-LMNA, pBABE-puro–GFP–progerin, shCTL-mLPx and mLPx-shZMPSTE24, were purchased from Addgene. The pInducer20-GATA4 and MSCV-GATA4 shRNA were provided by Dr. Stephen J. Elledge of Harvard medical school^{24 (link)}. Virus production and transduction were performed as previously described^{30 (link)}. Briefly, the retroviral plasmids pBABE-puro–control, pBABE-puro–GFP–wt-LMNA, pBABE-puro–GFP–progerin, shCTL-mLPx, mLPx-shZMPSTE24, and MSCV-GATA4 shRNA were transfected into 293FT cells with VSV-G and gag/pol plasmids using Fugene 6 transfection reagent (Roche, Basel, Switzerland). The viral supernatants were collected 48 and 72 h after transfection and were used to infect hUCB-MSCs in the presence of 5 μg/ml polybrene (Sigma). After the viral transduction, the hMSCs were selected with puromycin (0.5 μg/ml) for 4 days and cultured for 3 days before the analysis. The lentiviruses were generated according to the manufacturer (Thermo Fisher scientific, MA, USA) to introduce Dox-inducible flag-GATA4. The viral supernatants were collected 48 and 72 h after transfection and were used to infect hUCB-MSCs (MOI = 2) in the presence of 5 μg/ml polybrene (Sigma). At 24 h after transduction, the cells were washed four times in PBS and maintained in growth medium.