Department of Radiology, Molecular Imaging Program at Stanford, Stanford University, Stanford, CA 94305, USA.
Cell Transplant. 2012;21(12):2555-67. doi: 10.3727/096368912X653156. Epub 2012 Aug 2.
Magnetic resonance (MR) imaging of superparamagnetic iron oxide (SPIO)-labeled stem cells offers a noninvasive evaluation of stem cell engraftment in host organs. Excessive cellular iron load from SPIO labeling, however, impairs stem cell differentiation. The purpose of this study was to magnetically label human embryonic stem cells (hESCs) via a reduced exposure protocol that maintains a significant MR signal and no significant impairment to cellular pluripotency or differentiation potential. hESCs were labeled by simple incubation with Food and Drug Administration-approved ferumoxides, using concentrations of 50- 200 µg Fe/ml and incubation times of 3-24 h. The most reduced exposure labeling protocol that still provided a significant MR signal comparable to accepted labeling protocols was selected for subsequent studies. Labeled hESCs were compared to unlabeled controls for differences in pluripotency as studied by fluorescence staining for SSEA-1, SSEA-4, TRA-60, and TRA-81 and in differentiation capacity as studied by quantitative real-time PCR for hOCT4, hACTC1, hSOX1, and hAFP after differentiation into embryoid bodies (EBs). Subsequent MR and microscopy imaging were performed to evaluate for cellular iron distribution and long-term persistence of the label. An incubation concentration of 50 µg Fe/ml and incubation time of 3 h demonstrated a significantly reduced exposure protocol that yielded an intracellular iron uptake of 4.50 ± 0.27 pg, an iron content comparable to currently accepted SPIO labeling protocols. Labeled and unlabeled hESCs showed no difference in pluripotency or differentiation capacity. Ferumoxide-labeled hESCs demonstrated persistent MR contrast effects as embryoid bodies for 21 days. Electron microscopy confirmed persistent lysosomal storage of iron oxide particles in EBs up to 9 days, while additional microscopy visualization confirmed the iron distribution within single and multiple EBs. Labeling hESCs with ferumoxides by this tailored protocol reduces exposure of cells to the labeling agent while allowing for long-term visualization with MR imaging and the retention of cellular pluripotency and differentiation potential.
超顺磁性氧化铁 (SPIO) 标记的干细胞的磁共振 (MR) 成像是评估干细胞在宿主器官中定植的一种非侵入性方法。然而，SPIO 标记导致的细胞内铁负荷过多会损害干细胞的分化。本研究的目的是通过一种减少暴露的方案对人胚胎干细胞 (hESC) 进行磁性标记，该方案既能保持显著的 MR 信号，又不会显著损害细胞的多能性或分化潜能。通过简单地将美国食品和药物管理局批准的 Ferumoxides 与细胞孵育，使用 50-200μgFe/ml 的浓度和 3-24 小时的孵育时间来标记 hESC。选择提供与公认标记方案相当的显著 MR 信号的最减少暴露标记方案用于后续研究。通过荧光染色 SSEA-1、SSEA-4、TRA-60 和 TRA-81 研究多能性，通过定量实时 PCR 研究 hOCT4、hACTC1、hSOX1 和 hAFP 在分化为胚胎体 (EB) 后的分化能力，比较标记和未标记的 hESC 的差异。随后进行 MR 和显微镜成像以评估细胞内铁分布和标记的长期持久性。孵育浓度为 50μgFe/ml 和孵育时间为 3 小时的方案显示出一种明显减少暴露的方案，其细胞内铁摄取量为 4.50±0.27pg，铁含量与目前接受的 SPIO 标记方案相当。标记和未标记的 hESC 在多能性或分化能力上没有差异。作为胚胎体，Ferumoxides 标记的 hESC 显示出持续的 MR 对比效果长达 21 天。电子显微镜证实，氧化铁颗粒在 EB 中持续存在溶酶体储存，长达 9 天，而额外的显微镜可视化证实了铁在单个和多个 EB 中的分布。通过这种定制方案用 Ferumoxides 标记 hESC 可减少细胞暴露于标记剂的时间，同时允许通过 MR 成像进行长期可视化，并保留细胞的多能性和分化潜能。
