General Session: Innovative Technologies I - Hall F
Presented by: C. Ahuja
C. Ahuja(1,2), M. Khazaei(2), P. Chan(2), Y. Yao(2), Z. Lou(2), J. Bhavsar(2), J. Wang(2), M. Fehlings(1,2)
(1) Division of Neurosurgery, Department of Surgery, University of Toronto, Toronto, ON, Canada
(2) Department of Genetics and Development, Krembil Research Institute, Toronto Western Hospital, University Health Network, Toronto, ON, Canada
Introduction: Human induced pluripotent stem cell-derived neural stem cells (hiPS-NSCs) represent an exciting therapeutic strategy for traumatic spinal cord injury (SCI) as they can replace lost neural circuits, remyelinate denuded axons and provide local trophic support. Unfortunately, most individuals are in the chronic phase of their injury where dense perilesional chondroitin sulfate proteoglycan (CSPG) scarring significantly impairs neurite outgrowth and regenerative cell migration. Scar-modifying enzymes can enhance NSC-mediated recovery, however, nonspecific intrathecal administration produces off-target effects. We aimed to generate a genetically-engineered line of hiPS-NSCs, termed Spinal Microenvironment Modifying and Regenerative Therapeutic (SMaRT) cells, which are uniquely capable of expressing a scar-modifying enzyme within their local environment to enhance functional recovery.
Materials/Methods: Using non-viral transposon technology, a scar degrading enzyme was genetically integrated into hiPS-NSCs under a human promoter and a monoclonal line was generated by FACS (Fig-1A). Enzyme expression and activity was extensively characterized in vitro by biochemical assays, slot blot, and cell culture assays. T-cell deficient RNU rats with chronic (8 weeks; N=60) C6-7 clip-contusion injuries were randomized to receive (1) NSCs, (2) SMaRT enzyme-expressing NSCs, (3) vehicle, or (4) sham surgery. Behavioural assessments are ongoing; asynchronous rehabilitation will begin at 12 weeks post-transplant with a study endpoint of 32 weeks post-transplant. Weekly neurobehavioural assessments include BBB open-field locomotor scoring, inclined plane test, forelimb grip strength and tail flick sensory test. CatWalk digital gait assessment is being completed every 4 weeks.
Results: The scar-degrading ENZYME and fluorescent reporter are robustly expressed by transgenic SMaRT cells (Fig-1B). Importantly, SMaRT cells retain their human NSC characteristics (Fig-1C,D). The expressed enzyme rapidly degrades human CSPGs and allows neurons to extend into scar-mimicking CSPG-rich regions in vitro (Fig-1E). Conditioned SMaRT cell media also degrades rodent CSPGs in ex vivo injured cord cryosections. While blinded neurobehavioural assessments are ongoing, interim histologic analyses of several animals show that grafted human cells are extending remarkably long (≥20,000µm) axons along host white matter tracts rostrally and caudally. (Fig-2).
Conclusion: This work provides exciting proof-of-concept data that genetically-engineered SMaRT cells can degrade CSPGs in vitro and that human NSC transplants can grow long axons in chronic cervical SCI to potentially form a bridge for sensorimotor signal transmission. Figure 1. Generation and characterization of ENZYME-Expressing SMaRT hiPS-NSCs. (A) Vector design. (B) Ubiquitous transgene-reporter expression by SMaRT cells. (C) Neurosphere formation. (D) Retained capacity to differentiate to all three neuroglial lineages. (E) ENZYME expression by SMaRT cells allows growth into scar-like CSPG-dense regions. Figure 2. Transplanted human NSCs (GFP+) extend long axonal processes along host white matter tracts after chronic traumatic cSCI.