Gene Expression of Purified Beta-cell Tissue Obtained from Human Pancreas with Laser Capture Microdissection

Overview

Journal J Clin Endocrinol Metab

Publisher Oxford University Press

Specialty Endocrinology

Date 2007 Dec 13

PMID 18073315

Citations 41

Authors

Lorella Marselli

Jeffrey Thorne

Yu-Bae Ahn

Abdulkadir Omer

Dennis C Sgroi

Towia Libermann

Hasan H Otu

Arun Sharma

Susan Bonner-Weir

Gordon C Weir

Affiliations

Soon will be listed here.

Abstract

Context: 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.

Design: 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.

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

Citing Articles

Insulin regulates arginine-stimulated insulin secretion in humans.

Halperin F, Mezza T, Li P, Shirakawa J, Kulkarni R, Goldfine A Metabolism. 2022; 128:155117.

PMID: 34999111 PMC: 8821403. DOI: 10.1016/j.metabol.2021.155117.

A Systematic Comparison of Protocols for Recovery of High-Quality RNA from Human Islets Extracted by Laser Capture Microdissection.

Cefalo C, Mezza T, Giaccari A, Kulkarni R Biomolecules. 2021; 11(5).

PMID: 33922227 PMC: 8144988. DOI: 10.3390/biom11050625.

High Concentrations of Etanercept Reduce Human Islet Function and Integrity.

Brandhorst D, Brandhorst H, Acreman S, Abraham A, Johnson P J Inflamm Res. 2021; 14:599-610.

PMID: 33679137 PMC: 7926188. DOI: 10.2147/JIR.S294663.

Label-Free LC-MS/MS Strategy for Comprehensive Proteomic Profiling of Human Islets Collected Using Laser Capture Microdissection from Frozen Pancreata.

Zhang L, Lanzoni G, Battarra M, Inverardi L, Zhang Q Methods Mol Biol. 2018; 1871:253-264.

PMID: 30276744 PMC: 6317367. DOI: 10.1007/978-1-4939-8814-3_16.

Oxygen-permeable microwell device maintains islet mass and integrity during shipping.

Rojas-Canales D, Waibel M, Forget A, Penko D, Nitschke J, Harding F Endocr Connect. 2018; 7(3):490-503.

PMID: 29483160 PMC: 5861371. DOI: 10.1530/EC-17-0349.

References

Laybutt D, Kaneto H, Hasenkamp W, Grey S, Jonas J, Sgroi D . Increased expression of antioxidant and antiapoptotic genes in islets that may contribute to beta-cell survival during chronic hyperglycemia. Diabetes. 2002; 51(2):413-23. DOI: 10.2337/diabetes.51.2.413. View

Gregersen S, Thomsen J, Brock B, Hermansen K . Endothelin-1 stimulates insulin secretion by direct action on the islets of Langerhans in mice. Diabetologia. 1996; 39(9):1030-5. DOI: 10.1007/BF00400650. View

Van De Winkel M, Pipeleers D . Autofluorescence-activated cell sorting of pancreatic islet cells: purification of insulin-containing B-cells according to glucose-induced changes in cellular redox state. Biochem Biophys Res Commun. 1983; 114(2):835-42. DOI: 10.1016/0006-291x(83)90857-4. View

Balamurugan A, Bottino R, Giannoukakis N, Smetanka C . Prospective and challenges of islet transplantation for the therapy of autoimmune diabetes. Pancreas. 2006; 32(3):231-43. DOI: 10.1097/01.mpa.0000203961.16630.2f. View

Rakonczay Jr Z, Takacs T, Boros I, Lonovics J . Heat shock proteins and the pancreas. J Cell Physiol. 2003; 195(3):383-91. DOI: 10.1002/jcp.10268. View

Linetsky E, Bottino R, Lehmann R, Alejandro R, Inverardi L, Ricordi C . Improved human islet isolation using a new enzyme blend, liberase. Diabetes. 1997; 46(7):1120-3. DOI: 10.2337/diab.46.7.1120. View

Li C, Wong W . Model-based analysis of oligonucleotide arrays: expression index computation and outlier detection. Proc Natl Acad Sci U S A. 2001; 98(1):31-6. PMC: 14539. DOI: 10.1073/pnas.98.1.31. View

Van de Winkle M, Maes E, Pipeleers D . Islet cell analysis and purification by light scatter and autofluorescence. Biochem Biophys Res Commun. 1982; 107(2):525-32. DOI: 10.1016/0006-291x(82)91523-6. View

Cras-Meneur C, Inoue H, Zhou Y, Ohsugi M, Bernal-Mizrachi E, Pape D . An expression profile of human pancreatic islet mRNAs by Serial Analysis of Gene Expression (SAGE). Diabetologia. 2004; 47(2):284-99. DOI: 10.1007/s00125-003-1300-8. View

10.

Landgrebe J, Wurst W, Welzl G . Permutation-validated principal components analysis of microarray data. Genome Biol. 2002; 3(4):RESEARCH0019. PMC: 115254. DOI: 10.1186/gb-2002-3-4-research0019. View

11.

Hui H, Wang C, Li H, Bulotta A, DAmico E, Khoury N . Gene expression profiling of cultured human islet preparations. Diabetes Technol Ther. 2004; 6(4):481-92. DOI: 10.1089/1520915041705866. View

12.

Sander M, German M . The beta cell transcription factors and development of the pancreas. J Mol Med (Berl). 1997; 75(5):327-40. DOI: 10.1007/s001090050118. View

13.

Hori O, Miyazaki M, Tamatani T, Ozawa K, Takano K, Okabe M . Deletion of SERP1/RAMP4, a component of the endoplasmic reticulum (ER) translocation sites, leads to ER stress. Mol Cell Biol. 2006; 26(11):4257-67. PMC: 1489087. DOI: 10.1128/MCB.02055-05. View

14.

Lee T, Barshes N, Brunicardi F, Alejandro R, Ricordi C, Nguyen L . Procurement of the human pancreas for pancreatic islet transplantation. Transplantation. 2004; 78(3):481-3. DOI: 10.1097/01.tp.0000128910.41921.4b. View

15.

Ahmed M, Forsberg J, Bergsten P . Protein profiling of human pancreatic islets by two-dimensional gel electrophoresis and mass spectrometry. J Proteome Res. 2005; 4(3):931-40. DOI: 10.1021/pr050024a. View

16.

Schuit F, Flamez D, De Vos A, Pipeleers D . Glucose-regulated gene expression maintaining the glucose-responsive state of beta-cells. Diabetes. 2002; 51 Suppl 3:S326-32. DOI: 10.2337/diabetes.51.2007.s326. View

17.

Martinez A, Weaver C, Lopez J, Bhathena S, Elsasser T, Miller M . Regulation of insulin secretion and blood glucose metabolism by adrenomedullin. Endocrinology. 1996; 137(6):2626-32. DOI: 10.1210/endo.137.6.8641217. View

18.

Cnop M, Grupping A, Hoorens A, Bouwens L, Pipeleers-Marichal M, Pipeleers D . Endocytosis of low-density lipoprotein by human pancreatic beta cells and uptake in lipid-storing vesicles, which increase with age. Am J Pathol. 2000; 156(1):237-44. PMC: 1868647. DOI: 10.1016/s0002-9440(10)64724-4. View

19.

Emamaullee J, Shapiro A . Interventional strategies to prevent beta-cell apoptosis in islet transplantation. Diabetes. 2006; 55(7):1907-14. DOI: 10.2337/db05-1254. View

20.

Bottino R, Balamurugan A, Tse H, Thirunavukkarasu C, Ge X, Profozich J . Response of human islets to isolation stress and the effect of antioxidant treatment. Diabetes. 2004; 53(10):2559-68. DOI: 10.2337/diabetes.53.10.2559. View