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.