Gene expression of purified beta-cell tissue obtained from human pancreas with laser capture microdissection.
BACKGROUND
Human beta-cell gene profiling is a powerful tool for understanding beta-cell biology in normal and pathological conditions. Assessment is complicated when isolated islets are studied because of contamination by non-beta-cells and the trauma of the isolation procedure.


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


METHODS
Endogenous autofluorescence of beta-cells facilitated procurement of purified beta-cell tissue from frozen pancreatic sections with LCM. Gene expression profiles of three microdissected beta-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 beta-cell rich tissue in human pancreas. When compared with array profiles of purified beta-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.


CONCLUSIONS
LCM makes it possible to obtain beta-cell enriched tissue from human pancreas sections without the trauma and ischemia of islet isolation.
Marselli
Lorella
L
Section on Islet Transplantation and Cell Biology, Joslin Diabetes Center, One Joslin Place, Boston, Massachusetts 02215, USA.
Thorne
Jeffrey
J
Ahn
Yu-Bae
YB
Omer
Abdulkadir
A
Sgroi
Dennis C
DC
Libermann
Towia
T
Otu
Hasan H
HH
Sharma
Arun
A
Bonner-Weir
Susan
S
Weir
Gordon C
GC
D020869:Gene Expression Profiling; D006801:Humans; D050417:Insulin-Secreting Cells / Q000378:metabolism*; D007834:Lasers; D042282:Microdissection / Q000379:methods*; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D018384:Oxidative Stress; D012313:RNA / Q000032:analysis
D012313:RNA; D020928:Mitogen-Activated Protein Kinases