Isolating human islets of Langerhans causes loss of decay accelerating factor (CD55) on beta-cells.
It has previously been reported that human decay accelerating factor (DAF; CD55) is not expressed on cells isolated from human islets. We have investigated if this absence is caused by the islet isolation procedure and/or the single cell isolation technique. We focused on loss of DAF expression on beta-cells within the intact islet and on isolated individual beta-cells. We established that DAF was expressed in islets and on beta-cells prior to isolation by in situ analysis in the intact pancreas. In situ immunohistochemistry (IHC) was used to examine DAF expression on human pancreatic islets and isolated islets. A reverse transcriptase-polymerase chain reaction (RT-PCR) specific for human DAF mRNA was developed to measure mRNA levels in situ in islets within the intact pancreas, isolated islets, and purified beta-cells. beta-Cells were purified by fluorescence-activated cell sorting. DAF protein expression on these purified cells was measured using flow cytometry. Expression of DAF protein was present on the islets, including beta-cells within the human pancreas; however, comparative data from IHC and flow cytometry revealed the absence of DAF protein on beta-cells in both isolated islets and single cell preparations. Furthermore, compared to mRNA levels detected by in situ RT-PCR in the intact pancreas and in human HEK 293 cells, isolated islets, and purified human beta-cells showed downregulation of DAF mRNA. mRNA was detectable in both of these preparations by RT-PCR; levels were lower following both the islet isolation process (53%) and single cell preparation (a further 62%) compared to HEK 293 controls. Human islet allotransplantation might be more successful if either de novo transfer of DAF onto the isolated islets or novel techniques for islet isolation preserving DAF could be developed.
Immunology and Transplantation Research Group, Robarts Research Institute, University of Western Ontario, London, Ontario, Canada.
Joo Ho
JH
Tai
Hongtao
H
Sun
Weihua
W
Liu
C W James
CW
Melling
Craig
C
Hasilo
David J G
DJ
White
D000818:Animals; D018958:CD55 Antigens / Q000235:genetics*; D002460:Cell Line; D005786:Gene Expression Regulation; D006801:Humans; D050417:Insulin-Secreting Cells / Q000166:cytology* / Q000502:physiology / Q000637:transplantation*; D007515:Islets of Langerhans / Q000166:cytology* / Q000502:physiology; D007668:Kidney; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine
D018958:CD55 Antigens; D012333:RNA, Messenger