FIGURE 6.

How the type of cell labeling impacts on conclusions drawn from signals obtained from serial imaging. Cell viability can be assessed better from indirectly labeled cells than from directly labeled cells as shown by a cross-validation study using both direct and indirect cell labeling within the same cells. Reporter gene (luciferase and GFP)-expressing human embryonic stem cells (hES) or human embryonic stem cells differentiated to endothelial cells (hESC-EC) were directly labeled using iron oxide nanoparticles. (A) MRI imaging to track the directly loaded nanoparticle cell label (A/left) Serial in vivo MR [gradient-recalled echo (GRE)] images of iron oxide nanoparticles. No hypointense signal was found in control animals injected with unlabeled cells. MR signals showed no significant difference from day 2 to day 28 (the white arrow indicates teratoma formation in the hind limb injected with hES cells). (A/right) Quantitative analysis of GRE signals from all animals transplanted with hES cells and hESC-ECs [signal activity is expressed as authority unit (AU)]. (B) Tracking of the cells by virtue of reporter gene imaging (indirect cell labeling). (B/left) Planar bioluminescence imaging reveals differences in signals obtained from hind limbs that received either hES or hESC-EC cells. After initial similar signal decreases in both limbs, the signals from limbs with hES increased significantly over time, coinciding with teratoma formation in these limbs. (B/right) Quantification of 2D bioluminescence signals from each limb (photons/sec/cm²/sr; note the log10 scale). (C, top) Immunohistochemical (IHC) analysis of initially double labeled hES cells and hESC-ECs clearly reveals iron oxide (by Prussian Blue) co-localizing with a macrophage stain (by specific antibody Mac-3); IHC counterstains were Nuclear Fast Red and Hematoxylin, respectively. Note that macrophages loaded with iron particles can be found in between muscle bundles.(C, bottom) Immunofluorescence staining of GFP for transplanted luciferase co-expressing hESC-ECs (left) or hESC (right). Other panels show respective counterstains for microvasculature (CD31) or macrophages (Mac-3); nuclei were stained with DAPI (blue) in merged images. All images are from four weeks after transplantation. There were no transplanted GFP⁺ hESC-ECs found nearby macrophages. In tissues that received hES cells, GFP⁺ hESC were found to form teratoma (#) but no Prussian Blue-stained nanoparticles were found in corresponding IHC regions. The dashed line separates teratoma from normal muscle fibers (*). All scale bars are 20 μm. (Figure modified with permission from Li et al., 2008).