Detection and Characterization of Hepatic Engraftment of Embryonic Stem Derived Cells by Fluorescent Stereomicroscopy

Caballero, Montserrat; Lightfoot, Harry M.; LaPaglia, Michael; Pleasant, Andrew; Hatada, Seigo; Cairns, Bruce A.; Fair, Jeffrey H.

doi:10.1016/j.jss.2006.04.041

« Previous Next »Journal of Surgical Research
Volume 141, Issue 2 , Pages 134-140, August 2007

Detection and Characterization of Hepatic Engraftment of Embryonic Stem Derived Cells by Fluorescent Stereomicroscopy

Montserrat Caballero, Ph.D.
- Department of Surgery, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
- To whom correspondence and reprint requests should be addressed at Department of Surgery, University of North Carolina at Chapel Hill, 142 Glaxo Bldg., Chapel Hill, NC 27599-7211.
Harry M. Lightfoot Jr., M.D.
- Department of Surgery, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
Michael LaPaglia, M.D.
- Department of Surgery, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
Andrew Pleasant, B.S.
- Department of Surgery, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
Seigo Hatada, Ph.D.
- Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
Bruce A. Cairns, M.D.
- Department of Surgery, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
- These authors are co-senior authors for the article.
Jeffrey H. Fair, M.D.
- Department of Surgery, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
- These authors are co-senior authors for the article.

Received 27 September 2005 published online 02 June 2007.

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.

Material and 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.

Conclusion

Utilization of three-dimensional stereomicroscopy with a GFP2 long pass filter is a powerful and fast screening tool for GFP-ES derived hepatic engraftment.

Key Words: embryonic stem cells, liver, transplant, fluorescence, stereomicroscopy, engraftment efficiency