Detection and characterization of hepatic engraftment of embryonic stem derived cells by fluorescent stereomicroscopy.
BACKGROUND
Embryonic stem (ES) cells have been investigated as a potential replacement therapy for failed organs, such as the liver. However, detection of hepatic engraftment from candidate stem cells has been difficult due to low engraftment efficiency. Previous detection methods required that the graft be processed by molecular and/or immunohistochemical techniques, limiting further functional studies. This study evaluated the use of three-dimensional fluorescent stereomicroscopy for gross detection of ES cell derived hepatic engraftment.


METHODS
Murine ES cells expressing the enhanced green fluorescence protein (EGFP) underwent directed endodermal lineage differentiation. Three days after two thirds partial hepatectomy, cells were injected into the liver parenchyma, and livers were harvested at 10 to 20 d and examined by fluorescence stereomicroscopy with a GFP2 long pass filter (100447084; Leica Microsystems AG, Wetzlar, Germany). The sensitivity and reliability of the test was evaluated using quantitative polymerase chain reaction (q-PCR) to assay for the presence of EGFP mRNA in the tissue.


RESULTS
Fluorescent microscopy detected EGFP-positive cells engrafted with normal histology in 5 of 11 specimens. EGFP mRNA was confirmed in all five specimens by q-PCR. Only one of the 11 specimens was negative by fluorescence stereomicroscopy and positive by q-PCR, P < 0.02, Fisher's exact test.


CONCLUSIONS
Utilization of three-dimensional stereomicroscopy with a GFP2 long pass filter is a powerful and fast screening tool for GFP-ES derived hepatic engraftment.
Caballero
Montserrat
M
Department of Surgery, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7211, USA. montserrat_caballero@med.unc.edu
Lightfoot
Harry M
HM
Lapaglia
Michael
M
Pleasant
Andrew
A
Hatada
Seigo
S
Cairns
Bruce A
BA
Fair
Jeffrey H
JH
D000818:Animals; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000637:transplantation*; D005164:Factor IX / Q000096:biosynthesis; D049452:Green Fluorescent Proteins; D008099:Liver / Q000166:cytology / Q000601:surgery*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D016133:Polymerase Chain Reaction; D033581:Stem Cell Transplantation
D049452:Green Fluorescent Proteins; D005164:Factor IX