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A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation
Published on: April 2, 2012
Neural Progenitor Cells Expressing Herpes Simplex Virus-Thymidine Kinase for Ablation Have Differential
Zijian Lou1,2, Alexander Post1,2, Christopher E Rodgers1,2
1Division of Genetics and Development, Krembil Research Institute, University Health Network, Toronto, ON, Canada.
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.
Insights
Selective ablation of human neural progenitor cells (NPCs) after spinal cord injury (SCI) is crucial. Brivudine (BVDU) offers optimal ablation for loss-of-function studies with reduced bystander effects.
Area of Science:
- Neuroscience
- Regenerative Medicine
- Cell Therapy
Background:
- Neural progenitor cell (NPC) transplantation shows promise for spinal cord injury (SCI) recovery.
- Understanding the long-term function and contribution of grafted cells is essential.
- Selective cell ablation techniques can help elucidate the role of transplanted cells.
Purpose of the Study:
- To investigate the efficacy of brivudine (BVDU) for ablating human induced pluripotent stem cell-derived NPCs (iPSC-NPCs) expressing Herpes Simplex Virus Thymidine Kinase (HSV-TK).
- To evaluate the Herpes Simplex Virus Thymidine Kinase (HSV-TK) and ganciclovir (GCV)/brivudine (BVDU) system for in vitro and in vivo NPC ablation in a SCI model.
- To compare the ablation efficiency and bystander effects of GCV and BVDU.
Main Methods:
- Generation of HSV-TK-expressing human iPSC-NPCs.
- In vitro ablation assays using ganciclovir (GCV) and brivudine (BVDU).
- In vivo ablation studies in a translationally relevant spinal cord injury (SCI) model.
Main Results:
- Successful generation and in vitro ablation of iPSC-NPCs with >80% survival reduction.
- Validated synergistic HSV-TK and GCV/BVDU system for in vivo ablation.
- Combinatorial GCV and BVDU treatment enhanced ablation efficiency and reduced bystander effects.
- BVDU alone demonstrated optimal ablation for loss-of-function studies due to minimal nonselective cell killing.
Conclusions:
- The HSV-TK/prodrug system, particularly with BVDU, is effective for selective NPC ablation.
- BVDU is a promising agent for loss-of-function studies in the context of SCI regenerative therapies.
- This research provides a tool to better understand the contribution of grafted cells to functional recovery after SCI.
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