Engineering of Rhesus Monkey Pluripotent Stem Cells for Noninvasive Survey and Remote Control after Brain Allotransplantation

Abstract

Advanced technologies of targeted differentiation and genetic engineering in pluripotent stem cells (PSCs) offer production of purposefully designed cells for transplantation. Here, we engineer rhesus monkey PSCs (rhPSCs) to produce safe and functional neural progenitors and neurons, which could be noninvasively surveyed and controlled after brain transplantation. RhPSCs expressing hM3Dq-mCherry exhibited normal karyotype and had the ability to equally self-renew and differentiate into functional neurons as that of the mCherry expressing control. After allotransplantation into monkey prefrontal cortex, the hM3Dq-mCherry expressing rhPSC-derived cortical progenitors survived and matured gradually, which could be long-term surveyed by [18F]-Fluorodeoxyglucose positron emission tomography ([18F]FDG-PET) and electroencephalogram (EEG) following clozapine-N-oxide (CNO) administration. Remote activation of transplanted neurons caused increased [18F]-Fluorodeoxyglucose uptake started at 3 months and reached a plateau 9-12 months post-transplantation. EEG analysis revealed a decrease in the high delta spectrum power while an increase in the beta power after CNO delivery. Notably, no seizure-like spikes were observed even after repeated CNO exposure. Magnetic resonance (MR) imaging found no overgrowth in all allografts. Expression of herpes simplex virus thymidine kinase (HSVtk), a Ganciclovir (GCV)-induced suicide gene in rhPSCs further assured the safety without sacrificing the functional outcome of brain grafts. Our study offers a feasible strategy for long-term noninvasive survey and remote control of brain-grafted neurons.