P1 primary cell 4d nucleofector x kit

Apoptosis-deficient human MSCs were generated using Cas9 ribonucleoprotein (RNP) technology targeting BAK and BAX. The Cas9 RNP (Cas9 complexed with sgRNA targeting BAK, GGCCATGCTGGTAGACGTGT and BAX, TCTGACGGCAACTTCAACTG) (Synthego) was delivered using the P1 Primary Cell 4D-Nucleofector^TM X Kit (V4XP-1032, Lonza Bioscience) and 4D-Nucleofector^TM X Unit (Lonza Bioscience) following Amaxa^TM 4D-Nucleofector^TM protocol for undifferentiated human mesenchymal stem cells (Lonza Bioscience). Control MSCs were electroporated without sgRNA. Briefly, equal amount of sgRNA targeting BAK and BAX (1.2 pmol) were complexed with 104 pmol Cas9 enzyme in a 5 μL volume. 100,000 MSCs were resuspended in 15 μL Nucleofector™ and Supplement solution before adding to the Cas9 RNP complex to a total volume of 20uL in Nucleocuvette™. “FF104” programme in 4D-Nucleofector™ X Unit was used. After completion, prewarmed MSC medium was added to the electroporated cells before incubating for 10 mins in 37 °C, 5% CO₂ humidified incubator. After recovery, the cells were cultured and expanded in the same conditions as nontargeted MSCs. Apoptosis-resistant MSCs were selectively expanded following multiple rounds of treatment with BH3-mimetic drugs (1.25 μM x 2 rounds, then 10 μM x 2 rounds), as shown in Fig. 3A.

HASMCs were obtained from ScienCell (#6110) and cultured in smooth muscle cell medium (SMCM basal medium ScienCell, #1001), supplemented with 20% fetal bovine serum, 1% smooth muscle cell growth supplement, and 1% penicillin-streptomycin. HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 5% fetal bovine serum. Delivery of plasmids into HASMCs was performed via electroporation-based transfection (P1 Primary Cell 4D-Nucleofector X Kit, LONZA).