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Gene Expression of Purified β-Cell Tissue Obtained from Human Pancreas with Laser Capture Microdissection

Section on Islet Transplantation and Cell Biology (L.M., J.T., Y.-B.A., A.O., A.S., S.B.-W., G.C.W.), Research Division, Joslin Diabetes Center and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215; Molecular Pathology Unit (D.C.S.), Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114; and Beth Israel Deaconess Medical Center (T.L., H.H.O.), Harvard Medical School, Boston, Massachusetts 02215

Address all correspondence and requests for reprints to: Gordon C. Weir, M.D., Section on Islet Transplantation and Cell Biology, Joslin Diabetes Center, One Joslin Place, Boston, Massachusetts 02215. E-mail: gordon.weir{at}joslin.harvard.edu.

Context: Human β-cell gene profiling is a powerful tool for understanding β-cell biology in normal and pathological conditions. Assessment is complicated when isolated islets are studied because of contamination by non-β-cells and the trauma of the isolation procedure.

Objective: The objective was to use laser capture microdissection (LCM) of human β-cells from pancreases of cadaver donors and compare their gene expression with that of handpicked isolated islets.

Design: Endogenous autofluorescence of β-cells facilitated procurement of purified β-cell tissue from frozen pancreatic sections with LCM. Gene expression profiles of three microdissected β-cell samples and three isolated islet preparations were obtained. The array data were normalized using DNA-Chip Analyzer software (Harvard School of Public Health, Boston, MA), and the lower confidence bound evaluated differentially expressed genes. Real-time PCR was performed on selected acinar genes and on the duct cell markers, carbonic anhydrase II and keratin 19.

Results: Endogenous autofluorescence facilitates the microdissection of β-cell rich tissue in human pancreas. When compared with array profiles of purified β-cell tissue, with lower confidence bound set at 1.2, there were 4560 genes up-regulated and 1226 genes down-regulated in the isolated islets. Among the genes up-regulated in isolated islets were pancreatic acinar and duct genes, chemokine genes, and genes associated with hypoxia, apoptosis, and stress. Quantitative RT-PCR confirmed the differential expression of acinar gene transcripts and the duct marker carbonic anhydrase II in isolated islets.

Conclusion: LCM makes it possible to obtain β-cell enriched tissue from human pancreas sections without the trauma and ischemia of islet isolation.