Zaman Raiyan T, Tuerkcan Silvan, Mahmoudi Morteza, Saito Toshinobu, Yang Phillip C, Chin Frederick T, McConnell Michael V, Xing Lei
Department of Medicine, Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, CA, United States of America.
Department of Radiation Oncology, Division of Medical Physics, Stanford University School of Medicine, Stanford, CA, United States of America.
PLoS One. 2018 Feb 20;13(2):e0192662. doi: 10.1371/journal.pone.0192662. eCollection 2018.
Myocardial infarction (MI) causes significant loss of cardiomyocytes, myocardial tissue damage, and impairment of myocardial function. The inability of cardiomyocytes to proliferate prevents the heart from self-regeneration. The treatment for advanced heart failure following an MI is heart transplantation despite the limited availability of the organs. Thus, stem-cell-based cardiac therapies could ultimately prevent heart failure by repairing injured myocardium that reverses cardiomyocyte loss. However, stem-cell-based therapies lack understanding of the mechanisms behind a successful therapy, including difficulty tracking stem cells to provide information on cell migration, proliferation and differentiation. In this study, we have investigated the interaction between different types of stem and inflammatory cells and cell-targeted imaging molecules, 18F-FDG and 6-NBDG, to identify uptake patterns and pharmacokinetics in vitro.
Macrophages (both M1 and M2), human induced pluripotent stem cells (hiPSCs), and human amniotic mesenchymal stem cells (hAMSCs) were incubated with either 18F-FDG or 6-NBDG. Excess radiotracer and fluorescence were removed and a 100 μm-thin CdWO4 scintillator plate was placed on top of the cells for radioluminescence microscopy imaging of 18F-FDG uptake, while no scintillator was needed for fluorescence imaging of 6-NBDG uptake. Light produced following beta decay was imaged with a highly sensitive inverted microscope (LV200, Olympus) and an Electron Multiplying Charge-Couple Device (EM-CCD) camera. Custom-written software was developed in MATLAB for image processing.
The average cellular activity of 18F-FDG in a single cell of hAMSCs (0.670±0.028 fCi/μm2, P = 0.001) was 20% and 36% higher compared to uptake in hiPSCs (0.540±0.026 fCi/μm2, P = 0.003) and macrophages (0.430±0.023 fCi/μm2, P = 0.002), respectively. hAMSCs exhibited the slowest influx (0.210 min-1) but the fastest efflux (0.327 min-1) rate compared to the other tested cell lines for 18F-FDG. This cell line also has the highest phosphorylation but exhibited the lowest rate of de-phosphorylation. The uptake pattern for 6-NBDG was very different in these three cell lines. The average cellular activity of 6-NBDG in a single cell of macrophages (0.570±0.230 fM/μm2, P = 0.004) was 38% and 14% higher compared to hiPSCs (0.350±0.160 fM/μm2, P = 0.001) and hAMSCs (0.490±0.028 fM/μm2, P = 0.006), respectively. The influx (0.276 min-1), efflux (0.612 min-1), phosphorylation (0.269 min-1), and de-phosphorylation (0.049 min-1) rates were also highest for macrophages compared to the other two tested cell lines.
hAMSCs were found to be 2-3× more sensitive to 18F-FDG molecule compared to hiPSCs/macrophages. However, macrophages exhibited the most sensitivity towards 6-NBDG. Based on this result, hAMSCs targeted with 18F-FDG could be more suitable for understanding the mechanisms behind successful therapy for treating MI patients by gathering information on cell migration, proliferation and differentiation.
心肌梗死（MI）会导致心肌细胞大量损失、心肌组织损伤以及心肌功能受损。心肌细胞无法增殖使得心脏无法自我再生。尽管可供移植的器官有限，但MI后晚期心力衰竭的治疗方法仍是心脏移植。因此，基于干细胞的心脏治疗最终可能通过修复受损心肌来逆转心肌细胞损失，从而预防心力衰竭。然而，基于干细胞的治疗方法对成功治疗背后的机制缺乏了解，包括难以追踪干细胞以提供有关细胞迁移、增殖和分化的信息。在本研究中，我们研究了不同类型的干细胞与炎症细胞以及细胞靶向成像分子18F-FDG和6-NBDG之间的相互作用，以确定体外摄取模式和药代动力学。
将巨噬细胞（M1和M2）、人诱导多能干细胞（hiPSC）和人羊膜间充质干细胞（hAMSC）与18F-FDG或6-NBDG一起孵育。去除过量的放射性示踪剂和荧光物质，在细胞顶部放置一块100μm厚的钨酸镉闪烁体板，用于对18F-FDG摄取进行放射发光显微镜成像，而6-NBDG摄取的荧光成像则无需闪烁体。β衰变产生的光用高灵敏度倒置显微镜（LV200，奥林巴斯）和电子倍增电荷耦合器件（EM-CCD）相机成像。在MATLAB中开发了自定义软件用于图像处理。
hAMSC单个细胞中18F-FDG的平均细胞活性（0.670±0.028 fCi/μm2，P = 0.001）分别比hiPSC（0.540±0.026 fCi/μm2，P = 0.003）和巨噬细胞（0.430±0.023 fCi/μm2，P = 0.002）中的摄取高20%和36%。与其他测试细胞系相比，hAMSC对18F-FDG的流入速度最慢（0.210 min-1），但流出速度最快（0.327 min-1）。该细胞系的磷酸化水平也最高，但去磷酸化速率最低。这三种细胞系中6-NBDG的摄取模式非常不同。巨噬细胞单个细胞中6-NBDG的平均细胞活性（0.570±0.230 fM/μm2，P = 0.004）分别比hiPSC（0.350±0.160 fM/μm2，P = 0.001）和hAMSC（0.490±0.028 fM/μm2，P = 0.006）高38%和14%。与其他两个测试细胞系相比，巨噬细胞的流入（0.276 min-1）、流出（0.612 min-1）、磷酸化（0.269 min-1）和去磷酸化（0.049 min-1）速率也最高。
发现hAMSC对18F-FDG分子的敏感性比hiPSC/巨噬细胞高2 - 3倍。然而，巨噬细胞对6-NBDG表现出最高的敏感性。基于此结果，用18F-FDG靶向的hAMSC可能更适合通过收集有关细胞迁移、增殖和分化的信息来了解治疗MI患者成功治疗背后的机制。
