Ultrasound Control of Gene Expression in Human iPSCs via Heat Shock Promoters

Inducible systems are crucial tools in biomedical research, offering researchers spatiotemporal control at the cellular level. A promising development in this field is the use of focused ultrasound for controlling gene expression using heat shock promoters (HSPs). Focused ultrasound-induced mild hyperthermia activates the cellular heat shock response, which in turn activates HSPs and subsequently drives gene expression. Here, we utilized a Cre/LoxP system where each HSP drives Cre expression to investigate inducible gene expression with HSPs. Cre-mediated excision at the AAVS1 knock-in cassette results in constitutive expression of GFP. We assessed the performance of six HSPs in human induced pluripotent stem cells (hiPSCs). HSP16F and synHSPB'3 were the most effective, showing 27.7% and 33.5% GFP positivity, respectively, following 1 h of pulsed 42°C incubations. This contrasts with 0.6% and 3.5% GFP positivity at 37°C, indicating 45.9- and 9.7-fold increases, respectively. Increasing the number of HSP-Cre transposons did not significantly affect HSP16F but did enhance synHSPB'3, demonstrating its tunability. We then applied focused ultrasound to elevate the temperature to 42°C, resulting in 18.6% and 45.6% GFP positivity for HSP16F and synHSPB'3, respectively, compared to 0.3% and 6.2% at 37°C. Our design requires only a single, brief heat shock treatment to achieve permanent gene expression, enhancing its safety and feasibility for in vivo applications.