Abstract：
OBJECTIVE: The objective of this study was to develop and evaluate the feasibility and safety of a novel transaxial surgical approach for the delivery of human induced pluripotent stem cell-derived dopaminergic neuroprogenitor cells (DANPCs) into the putamen nucleus using nonhuman primates and surgical techniques and tools relevant to human clinical translation.
METHODS: Nine immunosuppressed, unlesioned adult cynomolgus macaques (4 females, 5 males) received intraputaminal injections of vehicle or DANPCs (0.9 × 105 to 1.1 × 105 cells/µL) under real-time intraoperative MRI guidance. The infusates were combined with 1-mM gadoteridol (for intraoperative MRI visualization) and delivered via two tracks per hemisphere (ventral and dorsal) using a transaxial approach. The total volumes of infusion were 25 µL and 50 µL for the right and left putamen, respectively (infusion rate 2.5 µL/min). Animals were evaluated with a battery of clinical and behavioral outcome measures and euthanized 7 or 30 days postsurgery; full necropsies were performed by a board-certified veterinary pathologist. Brain tissues were collected and processed for immunohistochemistry, including against the human-specific marker STEM121.
RESULTS: The optimized surgical technique and tools produced successful targeting of the putamen via the transaxial approach. Intraoperative MR images confirmed on-target intraputaminal injections in all animals. All animals survived to scheduled termination without clinical evidence of neurological deficits. The first 4 animals to undergo surgery had mild brain swelling noted at the end of surgery, of which 3 had transient reduced vision; administration of mannitol therapy and reduced intravenous fluid during the surgical procedure addressed these complications. Immunostaining against STEM121 confirmed the presence of grafted cells along the injection track within the targeted putamen area of DANPC-treated animals. All adverse histological findings were limited in scope and consistent with surgical manipulation, injection procedure, and postsurgical inflammatory response to the mechanical disruption caused by the cannula insertion.
CONCLUSIONS: The delivery system, injection procedure, and DANPCs were well tolerated in all animals. Prevention of mild brain swelling by mannitol dosing and reduction of intravenous fluids during surgery allowed visual effects to be avoided. The results of the study established that this novel transaxial approach can be used to correctly and safely target cell injections to the postcommissural putamen and support clinical investigation.
METHODS: Nine immunosuppressed, unlesioned adult cynomolgus macaques (4 females, 5 males) received intraputaminal injections of vehicle or DANPCs (0.9 × 105 to 1.1 × 105 cells/µL) under real-time intraoperative MRI guidance. The infusates were combined with 1-mM gadoteridol (for intraoperative MRI visualization) and delivered via two tracks per hemisphere (ventral and dorsal) using a transaxial approach. The total volumes of infusion were 25 µL and 50 µL for the right and left putamen, respectively (infusion rate 2.5 µL/min). Animals were evaluated with a battery of clinical and behavioral outcome measures and euthanized 7 or 30 days postsurgery; full necropsies were performed by a board-certified veterinary pathologist. Brain tissues were collected and processed for immunohistochemistry, including against the human-specific marker STEM121.
RESULTS: The optimized surgical technique and tools produced successful targeting of the putamen via the transaxial approach. Intraoperative MR images confirmed on-target intraputaminal injections in all animals. All animals survived to scheduled termination without clinical evidence of neurological deficits. The first 4 animals to undergo surgery had mild brain swelling noted at the end of surgery, of which 3 had transient reduced vision; administration of mannitol therapy and reduced intravenous fluid during the surgical procedure addressed these complications. Immunostaining against STEM121 confirmed the presence of grafted cells along the injection track within the targeted putamen area of DANPC-treated animals. All adverse histological findings were limited in scope and consistent with surgical manipulation, injection procedure, and postsurgical inflammatory response to the mechanical disruption caused by the cannula insertion.
CONCLUSIONS: The delivery system, injection procedure, and DANPCs were well tolerated in all animals. Prevention of mild brain swelling by mannitol dosing and reduction of intravenous fluids during surgery allowed visual effects to be avoided. The results of the study established that this novel transaxial approach can be used to correctly and safely target cell injections to the postcommissural putamen and support clinical investigation.
摘要：
目的：本研究的目的是开发和评估一种新型的经轴手术方法的可行性和安全性，该方法用于使用非人灵长类动物和与人类临床翻译相关的手术技术和工具将人诱导性多能干细胞衍生的多巴胺能神经祖细胞（DANPCs）传递到壳核中。
方法：九种免疫抑制，未释放的成年食蟹猴（4只雌性，5名男性）在实时术中MRI指导下接受了媒介物或DANPC（0.9×105至1.1×105细胞/µL）的静脉内注射。将输注液与1-mMgadoteridol（用于术中MRI可视化）结合，并使用经轴入路通过每个半球的两个轨道（腹侧和背侧）输送。左右壳核的输注总体积分别为25微升和50微升,分别(输注速率2.5微升/分钟)。用一系列临床和行为结果测量评价动物,并在手术后7或30天安乐死;由董事会认证的兽医病理学家进行完整的尸检。收集脑组织并进行免疫组化处理，包括针对人类特异性标记STEM121。
结果：优化的手术技术和工具通过经轴入路成功靶向壳核。术中MR图像证实了所有动物的目标内注射。所有动物存活至预定终止,没有神经缺陷的临床证据。前4只接受手术的动物在手术结束时出现轻度脑肿胀，其中3例出现短暂性视力下降;在手术过程中给予甘露醇治疗和减少静脉输液可解决这些并发症.针对STEM121的免疫染色证实了在DANPC处理的动物的靶向壳核区域内沿着注射轨迹存在移植细胞。所有不良组织学发现范围有限，与手术操作一致。注射程序,以及由插管插入引起的机械破坏的术后炎症反应。
结论：输送系统，注射程序,和DANPCs在所有动物中均有良好的耐受性。通过甘露醇给药和减少手术期间的静脉输液来预防轻度脑肿胀可以避免视觉效果。研究结果确定，这种新颖的跨轴方法可用于正确，安全地将细胞注射到连合后壳核并支持临床研究。
方法：九种免疫抑制，未释放的成年食蟹猴（4只雌性，5名男性）在实时术中MRI指导下接受了媒介物或DANPC（0.9×105至1.1×105细胞/µL）的静脉内注射。将输注液与1-mMgadoteridol（用于术中MRI可视化）结合，并使用经轴入路通过每个半球的两个轨道（腹侧和背侧）输送。左右壳核的输注总体积分别为25微升和50微升,分别(输注速率2.5微升/分钟)。用一系列临床和行为结果测量评价动物,并在手术后7或30天安乐死;由董事会认证的兽医病理学家进行完整的尸检。收集脑组织并进行免疫组化处理，包括针对人类特异性标记STEM121。
结果：优化的手术技术和工具通过经轴入路成功靶向壳核。术中MR图像证实了所有动物的目标内注射。所有动物存活至预定终止,没有神经缺陷的临床证据。前4只接受手术的动物在手术结束时出现轻度脑肿胀，其中3例出现短暂性视力下降;在手术过程中给予甘露醇治疗和减少静脉输液可解决这些并发症.针对STEM121的免疫染色证实了在DANPC处理的动物的靶向壳核区域内沿着注射轨迹存在移植细胞。所有不良组织学发现范围有限，与手术操作一致。注射程序,以及由插管插入引起的机械破坏的术后炎症反应。
结论：输送系统，注射程序,和DANPCs在所有动物中均有良好的耐受性。通过甘露醇给药和减少手术期间的静脉输液来预防轻度脑肿胀可以避免视觉效果。研究结果确定，这种新颖的跨轴方法可用于正确，安全地将细胞注射到连合后壳核并支持临床研究。
