Neural Progenitor Cells Expressing Herpes Simplex Virus-Thymidine Kinase for Ablation Have Differential Chemosensitivity to Brivudine and Ganciclovir.

Zijian Lou,Mahmood Chamankhah,James Hong,Michael G Fehlings,Christopher S Ahuja,Christopher E Rodgers,Alexander Post,Mohamad Khazaei

doi:10.3389/fncel.2021.638021

Abstract

Neural progenitor cell (NPC) transplants are a promising therapy for treating spinal cord injury (SCI), however, their long-term role after engraftment and the relative contribution to ongoing functional recovery remains a key knowledge gap. Selective human cell ablation techniques, currently being developed to improve the safety of progenitor cell transplant therapies in patients, may also be used as tools to probe the regenerative effects attributable to individual grafted cell populations. The Herpes Simplex Virus Thymidine Kinase (HSV-TK) and ganciclovir (GCV) system has been extensively studied in the context of SCI and broader CNS disease. However, the efficacy of brivudine (BVDU), another HSV-TK prodrug with potentially reduced bystander cytotoxic effects and in vivo toxicity, has yet to be investigated for NPC ablation. In this study, we demonstrate successful generation and in vitro ablation of HSV-TK-expressing human iPSC-derived NPCs with a >80% reduction in survival over controls. We validated an HSV-TK and GCV/BVDU synergistic system with iPSC-NPCs using an efficient gene-transfer method and in vivo ablation in a translationally relevant model of SCI. Our findings demonstrate enhanced ablation efficiency and reduced bystander effects when targeting all rapidly dividing cells with combinatorial GCV and BVDU treatment. However, for use in loss of function studies, BVDU alone is optimal due to reduced nonselective cell ablation.

Highlights

Neural progenitor cell (NPC) transplants are an exciting therapy for numerous neurodegenerative conditions including traumatic spinal cord injury (SCI; Ahuja et al, 2017a,b)
We evaluated the cell-killing efficiency of both the GCV and BVDU prodrugs on herpes simplex virus-thymidine kinase (HSV-TK)+ NPCs, via DAPI staining of the total attached cells remaining after various timepoints and concentrations of GCV/BVDU treatment (Figure 2 and Supplementary Figure 1)
The remaining numbers of HSV-TK NPCs in each well were normalized to the 0 drug control of that time point to account for cell proliferation after the

Summary

Introduction

Neural progenitor cell (NPC) transplants are an exciting therapy for numerous neurodegenerative conditions including traumatic spinal cord injury (SCI; Ahuja et al, 2017a,b). There is evidence that NPC transplants lead to improved functional recovery, the roles of the differentiated cells post-engraftment and the mechanisms by which they enhance regeneration of the spinal cord remains a key knowledge gap. The exogenous protein can catalyze the conversion of a prodrug into a cytotoxic compound, ablating transfected tumor cells. These systems have been studied for use in transplant therapies, providing a failsafe mechanism for cells in the event that they form teratomas (Jones et al, 2014; Greco et al, 2015; Yagyu et al, 2015; Liang et al, 2018; Kojima et al, 2019). The selectivity and effectiveness of these suicide-gene systems can be used to elucidate the regenerative effects associated with individual cell populations originating from the graft post-transplantation through loss of function experiments

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Conclusion