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Table 4 Comparison of contemporary cell tracking methods

From: Human adipose-derived stromal/stem cells demonstrate short-lived persistence after implantation in both an immunocompetent and an immunocompromised murine model

Tracking method – mechanism Disadvantages Advantages
GFP, luciferase – DNA transfer can be mediated virally (transduction), via liposomes (lipofection), or by electrical parameters (electroporation/transfection) - Genetic manipulation of cells may alter their function - Human ASCs with GFP or luciferase resume proliferation normally
- Since they are cytosolic in location; the vector (mRNA/DNA) and/or the protein (GFP) could be transmitted to host cells via fusion elements and/or microvesicle secretion, resulting in contamination. Hence, there is concern for false positivity and their detection may not equate to viability of donor cells - Detection is sensitive to in vivo non-invasive bioluminescence imaging
- Technique requires serial passages, not suitable for use with fresh uncultured cells  
- Many mammalian tissues have endogenous fluorescence  
BrdU – this nuclear marker is a thymidine analog that replaces (3H) thymidine and can penetrate cell membranes to incorporate into newly synthesized DNA strands of actively proliferating cells - Optimal labeling requires longer incubation time - BrdU labeling has no effects on the ASC differentiation/proliferation and is not cytotoxic
- Not suitable for non-invasive methods of detection
- Does not indicate viability
- Cells lose BrdU rapidly with serial passages
Lipophilic dyes (DiI, DiR) –long-chain carbocyanine dyes with long aliphatic tails that incorporate into the lipid regions of the cell membranes - Rapidly contaminates neighboring cells by macrophage-mediated phagocytosis, exchange of membrane microdomains, microvesicle and/or exosome transfer - Easy technique for labeling and identification of cells
- May be cytotoxic to cells
- The dye fades with serial passages
Amine reactive probes (CFSE) – these diffuse into cells and react with cytosolic amine-containing residues to form dye–protein adducts that are retained - The dye is toxic to ASCs - Good staining efficiency
- Not suitable for in vivo non-invasive imaging
- Does not correlate with viability
- Contamination of neighboring cells can occur via macrophage-mediated phagocytosis, microvesicle and/or exosome transfer
Nanoparticles – small crystals made up of inorganic molecules; for example, iron oxide, cadmium - Can be toxic to cells in high concentration and detection is difficult with low concentrations - Photostable, remain resistant for long periods of time
- Contamination of neighboring cells can occur via phagocytosis, microvesicles and/or exosome transfer - Can be used for in vivo non-invasive imaging
- Does not correlate with viability
Real-time PCR for endogenous retroviral sequence (ERV-3) – the gene is present as a single copy in the human genome and so can be used to detect the presence of transplanted human cells in animal models - Not suitable for non-invasive methods of detection - Gives a quantitative estimate of number of cells
- False positives can occur by macrophage-mediated phagocytosis but this is very low - Very sensitive and specific
  - The gene is already present in the human cells, so there is no need to stain the cells
FISH detection of human-specific cell surface markers or Alu sequences - Alu sequences occur in large numbers in the primate genome, which makes higher likelihood of a false positive detection by transmission to host cells/macrophages via microvesicles/exosomes - The gene is already present in the human cells, so there is no need to stain the cells
- Not suitable for non-invasive methods of detection  
- Does not correlate with viability  
  1. ASC, adipose-derived stromal/stem cell; BrdU, 5-Bromo-2-deoxyurudine; CFSE, carboxyfluorescein succinimidyl ester; ERV-3, endogenous retrovirus 3; FISH, fluorescent in situ hybridization; GFP, green fluorescent protein.