title: Dynamic tracking of stem cells in an acute liver failure model.
abstract: OBJECTIVE
  To investigate a dual labeling technique, which would enable real-time monitoring of transplanted embryonic stem cell (ESC) kinetics, as well as long-term tracking.
  
  
  METHODS
  Liver damage was induced in C57/BL6 male mice (n = 40) by acetaminophen (APAP) 300 mg/kg administered intraperitoneally. Green fluorescence protein (GFP) positive C57/BL6 mouse ESCs were stained with the near-infrared fluorescent lipophilic tracer 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR) immediately before transplantation into the spleen. Each of the animals in the cell therapy group (n = 20) received 5 × 10(6) ESCs 4 h following treatment with APAP. The control group (n = 20) received the vehicle only. The distribution and dynamics of the cells were monitored in real-time with the IVIS Lumina-2 at 30 min post transplantation, then at 3, 12, 24, 48 and 72 h, and after one and 2 wk. Immunohistochemical examination of liver tissue was used to identify expression of GFP and albumin. Plasma alanine aminotransferase (ALT) was measured as an indication of liver damage.
  
  
  RESULTS
  DiR-stained ESCs were easily tracked with the IVIS using the indocyanine green filter due to its high background passband with minimal background autofluorescence. The transplanted cells were confined inside the spleen at 30 min post-transplantation, gradually moved into the splenic vein, and were detectable in parts of the liver at the 3 h time-point. Within 24 h of transplantation, homing of almost 90% of cells was confirmed in the liver. On day three, however, the DiR signal started to fade out, and ex vivo IVIS imaging of different organs allowed signal detection at time-points when the signal could not be detected by in vivo imaging, and confirmed that the highest photon emission was in the liver (P < 0.0001). At 2 wk, the DiRsignal was no longer detectable in vivo; however, immunohistochemistry analysis of constitutively-expressed GFP was used to provide an insight into the distribution of the cells. GFP +ve cells were detected in tissue sections resembling hepatocytes and were dispersed throughout the hepatic parenchyma, with the presence of a larger number of GFP +ve cells incorporated within the sinusoidal endothelial lining. Very faint albumin expression was detected in the transplanted GFP +ve cells at 72 h; however at 2 wk, few cells that were positive for GFP were also strongly positive for albumin. There was a significant improvement in serum levels of ALT, albumin and bilirubin in both groups at 2 wk when compared with the 72 h time-point. In the cell therapy group, serum ALT was significantly (P = 0.016) lower and albumin (P = 0.009) was significantly higher when compared with the control group at the 2 wk time-point; however there was no difference in mortality between the two groups.
  
  
  CONCLUSIONS
  Dual labeling is an easy to use and cheap method for longitudinal monitoring of distribution, survival and engraftment of transplanted cells, and could be used for cell therapy models.
authors.affiliation: HPB and Liver Transplantation Surgery, Royal Free Hospital, University College London, London NW3 2PF, UK.
authors.forename: Tarek
authors.initials: T
authors.lastname: Ezzat
authors.forename: Dipok Kumar
authors.initials: DK
authors.lastname: Dhar
authors.forename: Massimo
authors.initials: M
authors.lastname: Malago
authors.forename: Steven W M
authors.initials: SW
authors.lastname: Olde Damink
mesh_terms: D000082:Acetaminophen / Q000633:toxicity; D018712:Analgesics, Non-Narcotic / Q000633:toxicity; D000818:Animals; D002232:Carbocyanines / Q000378:metabolism; D002478:Cells, Cultured; D004195:Disease Models, Animal; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D017114:Liver Failure, Acute / Q000139:chemically induced / Q000503:physiopathology / Q000628:therapy*; D008111:Liver Function Tests; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D013194:Staining and Labeling / Q000379:methods*; D033581:Stem Cell Transplantation / Q000379:methods*
chemical_list: D018712:Analgesics, Non-Narcotic; D002232:Carbocyanines; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D000082:Acetaminophen