Development and Characterization of Self-Tracing Neural Progenitor Cells for Mapping Their Synaptic Integration into Endogenous Neural Networks

Abstract

Neural progenitor cell (NPC) transplantation holds immense promise for neurodegenerative and traumatic central nervous system (CNS) pathologies. However, it is crucial to define which neural circuits and pathways are targeted with transplanted NPCs under different conditions. A major roadblock lies in the limited ability to accurately trace integration of grafted cells into the host neural network. Conventional tracers suffer from drawbacks like low trans-synaptic efficiency, toxicity, and difficulty in efficiently and specifically targeting transplanted cells. To address these critical limitations, we have developed self-tracing NPCs genetically engineered to express both anterograde (WGA-mCherry) and retrograde (GFP-TTC) trans-synaptic tracers. These self-tracing NPCs maintain their intrinsic properties, differentiate into electrically active neurons, and integrate into host circuitry in vitro. Importantly, co-culture with primary rat neurons revealed successful trans-synaptic tracing of grafted human neurons, evidenced by single-positive WGA+ or TTC+ rat cells. In vivo, NPCs transplanted into a rodent spinal cord injury model retained tracer expression for 12 weeks, enabling visualization of grafted cells within the spinal cord. Co-labeling with WGA and TTC provided evidence that these NPCs forms neurons which integrated into local circuits. Our novel self-tracing NPC platform offers a powerful tool to overcome trans-synaptic tracing challenges. This approach provides the opportunity to gain critical insights into graft integration and neural circuit remodeling, paving the way for better-designed transplantation strategies and improved therapeutic outcomes in a broad spectrum of CNS disorders.