From iPSCs to Hematopoietic Lineage: An Endoribonuclease Mediated Cell-Type-Specific Genetic System to Guide Automated Differentiation

hiPSCs provide unprecedented opportunities for cell therapies against intractable diseases. To produce therapeutic immune cells, recent advances emphasize the inevitable role of endothelial-to-hematopoietic transition (EHT), but still face a significant challenge in building a genetic program for cell-type-specific differentiation. To tackle this challenge, we built a novel platform for genetic programming, where endoribonucleases from the CRISPR family mediate sensing of endogenous cell-type-specific microRNA (miR) and actuate cell differentiation by transcriptional factor (TF) overexpression. We applied this system to differentiate hiPSC into hematopoietic lineages with an intermediate endothelial state. First, we extensively searched databases to identify endothelial-specific miRs and TFs that are critical for EHT. We designed and built high miR sensors and optimized the circuit elements’ stoichiometries by a poly-transfection method and achieved a fold change as high as 63. Then we verified the cell type specificity of this sensor using endothelial and non-endothelial cell lines. We monitored the temporal dynamics of endogenous miR during cell differentiation using a programmed differentiation from hiPSCs to endothelial cells via a 4-day protocol. With these solid validations, we replaced the reporter of the high miR sensor with TFs to guide differentiation to the hematopoietic lineage. Our results showed that the miR sensing and TFs actuating systems can derive significant percentages of CD34+CD45+ hematopoietic-like cells from hiPSCs. Together, this genetic system will provide a novel platform to program cell-type-specific differentiation, which will provide insights into the universal generation of functional and therapeutic immune cells from unlimited and reprogrammable hiPSC sources.