Magnetized Cellbots to Spatiotemporally Control Differentiation of Human-Induced Pluripotent Stem Cells

Precise spatiotemporal control of gene expression and cellular differentiation is essential for engineering native-like multicellular structures. Current cell differentiation approaches typically rely on externally provided inputs whose effects are not targeted to distinct cells in the appropriate state and hence cannot spatially organize and mature tissue structures as needed. Our work introduces a magnetically controlled microrobot (MR) platform for guiding mammalian cells to desired locations that, combined with synthetic biology, delivers biological signals at precise locations and times, enabling spatiotemporal control of cell-fate decision-making. We use synNotch, a cell-cell contact-based biological signaling that induces relevant gene expression in receivers when the receiver cells contact sender cells through ligand-receptor binding. Magnetically driven MRs are then allowed to be internalized by sender cells, resulting in magnetized sender cellbots. Using a 3-pair orthogonal Helmholtz coil system, we guided magnetized sender cellbots to precise locations in a receiver cell culture, activating desired fluorescent protein expression in target Chinese Hamster Ovary (CHO) receiver cells. Next, we engineered Human-Induced Pluripotent Stem Cells (hiPSC) to function as receivers that can be instructed by senders to differentiate into endothelial cells (ECs) via overexpression of ETV2 (ETS variant transcription factor 2), a master transcriptional regulator of endothelial cell development. Using our magnetic platform, we guided multiple sender cellbots to target locations on a monolayer of hiPSC receivers, resulting in differentiation of receivers into ECs and possible onset of vascular formation. Our approach provides a foundation for the engineering spatial patterns by activating conditional triggers based on MR location and cell state at multiple time points, enabling several applications such as control of organoid architecture.