Date Published: February 20, 2018
Publisher: Public Library of Science
Author(s): Raiyan T. Zaman, Silvan Tuerkcan, Morteza Mahmoudi, Toshinobu Saito, Phillip C. Yang, Frederick T. Chin, Michael V. McConnell, Lei Xing, Chin-Tu Chen.
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.
Myocardial infarction (MI), one of the leading causes of death in the United States and worldwide, results in significant cardiomyocyte loss, myocardial tissue damage, and impairment of myocardial function [1, 2]. Cardiomyocyte loss due to MI injury is considered irreversible, with the heart lacking sufficient capacity for self-regeneration . Cell-based cardiac therapies are considered an attractive therapeutic alternative to reverse cardiomyocyte loss by repairing the injured myocardium that would ultimately prevent heart failure . To date, a wide variety of cell sources, both of adult and embryonic origin, have been investigated for use in heart repair, with mixed outcomes [5, 6]. Current treatments of myocardial injury only slow down the disease progression without facilitating any myocardial repair . The limited mitotic capacity of cardiac cells and decreased cellularity after injury leads to suboptimal natural regenerative potential of myocardial tissue. The treatment for advanced heart failure following an MI is heart transplantation despite the limited availability of the organs. . Therefore, multiple pre-clinical researches specifically employed embryonic stem cells (ESCs) to restore the heart function by regenerating myocardial tissue [9–18]. Although, these outcomes inspired numerous cell-therapy-based clinical trials, it has been difficult to obtain unequivocal evidence for robust clinical benefit . Thus, there is a great need to understand the mechanisms behind a successful therapy through tracking stem cell migration, proliferation, and differentiation in vivo using targeted imaging molecules. In this study, we successfully investigated three different cell lines—macrophages, human induced pluripotent stem cells (hiPSCs), and human amniotic mesenchymal stem cells (hAMSCs) and their interaction pattern with two different targeted imaging molecules [18F]fluoro-deoxyglucose (18F-FDG) and 6-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-6-Deoxyglucose (6-NBDG). A newly developed Radioluminescence Microscopy technique was used to gather the pharmacokinetic information . This pharmacokinetic information can be useful in decision making for the specific cell selection process and corresponding imaging molecule to enable tracking therapeutic cells for their migration, proliferation and differentiation patterns in myocardial repair.
We observed good co-localization between the radioluminescence and fluorescence intensity and the cell outline seen on brightfield images based on the acquired brightfield, radioluminescence and fluorescence micrographs (Figs 3A and 4A). Although there were significant variance identified in the average uptake of 18F-FDG and 6-NBDG for all three cell lines, the relationship between cell-to-cell comparisons was found to be linear for macrophages, hiPSCs, and hAMSCs.
In this study, to the best of our knowledge, we are the first to successfully investigate radioluminescence and fluorescence microscopy for detailed imaging and pharmacokinetic modeling, at the single cell level of inflammatory and stem cells and their uptake of targeted imaging molecules 18F-FDG and 6-NBDG. Based on our preliminary data, hAMSCs were found to be more sensitive towards 18F-FDG uptake compared to hiPSCs and macrophages. However, macrophages exhibited the most sensitivity to 6-NBDG uptake.
We found hAMSCs were 2–3× more sensitive to 18F-FDG uptake than macrophages, while macrophages exhibited the most sensitivity towards 6-NBDG uptake. Based on this result, hAMSCs targeted with 18F-FDG could be more suitable for understanding the mechanisms behind successful cell therapy for treating MI patients.