Haralampieva Deana, Betzel Thomas, Dinulovic Ivana, Salemi Souzan, Stoelting Meline, Krämer Stefanie D, Schibli Roger, Sulser Tullio, Handschin Christoph, Eberli Daniel, Ametamey Simon M
Institute of Pharmaceutical Sciences, ETH Zurich, Zurich, Switzerland Laboratory for Tissue Engineering and Stem Cell Therapy, Department of Urology, University Hospital Zurich, University of Zurich, Zurich, Switzerland Zurich Center for Integrative Human Physiology, Zurich, Switzerland; and.
Institute of Pharmaceutical Sciences, ETH Zurich, Zurich, Switzerland.
J Nucl Med. 2016 Sep;57(9):1467-73. doi: 10.2967/jnumed.115.170548. Epub 2016 May 19.
Transplantation of human muscle precursor cells (hMPCs) is envisioned for the treatment of various muscle diseases. However, a feasible noninvasive tool to monitor cell survival, migration, and integration into the host tissue is still missing.
In this study, we designed an adenoviral delivery system to genetically modify hMPCs to express a signaling-deficient form of human dopamine D2 receptor (hD2R). The gene expression levels of the receptor were evaluated by reverse transcriptase polymerase chain reaction, and infection efficiency was evaluated by fluorescent microscopy. The viability, proliferation, and differentiation capacity of the transduced cells, as well as their myogenic phenotype, were determined by flow cytometry analysis and fluorescent microscopy. (18)F-fallypride and (18)F-fluoromisonidazole, two well-established PET radioligands, were assessed for their potential to image engineered hMPCs in a mouse model and their uptakes were evaluated at different time points after cell inoculation in vivo. Biodistribution studies, autoradiography, and PET experiments were performed to determine the extent of signal specificity. To address feasibility for tracking hMPCs in an in vivo model, the safety of the adenoviral gene delivery was evaluated. Finally, the harvested tissues were histologically examined to determine whether survival of the transplanted cells was sustained at different time points.
Adenoviral gene delivery was shown to be safe, with no detrimental effects on the primary human cells. The viability, proliferation, and differentiation capacity of the transduced cells were confirmed, and flow cytometry analysis and fluorescent microscopy showed that their myogenic phenotype was sustained. (18)F-fallypride and (18)F-fluoromisonidazole were successfully synthesized. Specific binding of (18)F-fallypride to hD2R hMPCs was demonstrated in vitro and in vivo. Furthermore, the (18)F-fluoromisonidazole signal was high at the early stages. Finally, sustained survival of the transplanted cells at different time points was confirmed histologically, with formation of muscle tissue at the site of injection.
Our proposed use of a signaling-deficient hD2R as a potent reporter for in vivo hMPC PET tracking by (18)F-fallypride is a significant step toward potential noninvasive tracking of hD2R hMPCs and bioengineered muscle tissues in the clinic.
人们设想将人类肌肉前体细胞（hMPCs）移植用于治疗各种肌肉疾病。然而，目前仍缺少一种可行的非侵入性工具来监测细胞存活、迁移以及整合到宿主组织中的情况。
在本研究中，我们设计了一种腺病毒递送系统，对hMPCs进行基因改造，使其表达一种信号缺陷型的人类多巴胺D2受体（hD2R）。通过逆转录聚合酶链反应评估受体的基因表达水平，通过荧光显微镜评估感染效率。通过流式细胞术分析和荧光显微镜确定转导细胞的活力、增殖能力、分化能力及其成肌表型。评估了两种成熟的正电子发射断层扫描（PET）放射性配体（18）F-氟哌利多和（18）F-氟米索硝唑在小鼠模型中对工程化hMPCs成像的潜力，并在体内接种细胞后的不同时间点评估它们的摄取情况。进行生物分布研究、放射自显影和PET实验以确定信号特异性的程度。为了解决在体内模型中追踪hMPCs的可行性问题，评估了腺病毒基因递送的安全性。最后，对收获的组织进行组织学检查，以确定移植细胞在不同时间点是否持续存活。
腺病毒基因递送被证明是安全的，对原代人类细胞没有有害影响。证实了转导细胞的活力、增殖能力和分化能力，流式细胞术分析和荧光显微镜显示它们的成肌表型得以维持。成功合成了（18）F-氟哌利多和（18）F-氟米索硝唑。在体外和体内均证明了（18）F-氟哌利多与hD2R hMPCs的特异性结合。此外，（18）F-氟米索硝唑信号在早期阶段较高。最后，通过组织学证实了移植细胞在不同时间点的持续存活，在注射部位形成了肌肉组织。
我们提议使用信号缺陷型hD2R作为通过（18）F-氟哌利多在体内对hMPCs进行PET追踪的有效报告基因，这是朝着在临床上对hD2R hMPCs和生物工程肌肉组织进行潜在非侵入性追踪迈出的重要一步。
