Adaptive metal ion transport and metalloregulation-driven differentiation in pluripotent synthetic cells

Pluripotent cells can yield different cell types determined by the specific sequence of differentiation signals that they encounter as the cell activates or deactivates functions and retains memory of previous inputs. Here, we achieved pluripotency in synthetic cells by incorporating three dormant apo-metalloenzymes such that they could differentiate towards distinct fates, depending on the sequence of specific metal ion transport with ionophores. In the first differentiation step, we selectively transported one of three extracellular metal ion cofactors into pluripotent giant unilamellar vesicles (GUVs), which resulted in elevation of intracellular pH, hydrogen peroxide production or GUV lysis. Previously added ionophores suppress transport with subsequent ionophores owing to interactions among them in the membrane, as corroborated by atomistic simulations. Consequently, the addition of a second ionophore elicits a dampened response in the multipotent GUV and a third ionophore results in no further response, reminiscent of a terminally differentiated GUV. The pluripotent GUV can differentiate into five final fates, depending on the sequence in which the three ionophores are added. The sequence of specific differentiation signals determines the fate of a pluripotent cell. Here pluripotency was introduced into synthetic cells by loading them with three dormant apo-metalloenzymes, which were activated through selective metal ion transport by one of three ionophores. Depending on the sequence of metal ion intake, the synthetic cells differentiated towards five distinct fates.