Impact of integrin-matrix matching and inhibition of apoptosis on the survival of purified human beta-cells in vitro.
OBJECTIVE
Human islet cells survive poorly in culture and are overgrown by non-endocrine cells. The aims of this study were to sort human beta cells and to develop approaches for their improved survival in culture.


METHODS
Human islets were infected with recombinant adenovirus expressing green fluorescent protein (GFP) under the control of the rat insulin promoter such that only beta cells expressed GFP. GFP-positive beta cells were sorted by flow cytometry, and expression of select integrins evaluated by RT-PCR. Beta cells were cultured on different extracellular matrices for up to 15 days. Apoptosis was measured by annexin V binding and ELISA. Insulin secretion was measured by ELISA.


RESULTS
Sorted beta cells survived less well in culture than unsorted islet cells. This did not appear to be due to adenoviral infection and/or GFP expression. Purified beta cells expressed the integrins alpha 3, alpha 5, alpha 6, alpha V, beta1, but not beta 4. Of the various matrices tested, sorted beta cells attached and spread best on a lawn of lysed human bladder carcinoma cells (5637 cells). However, survival remained poor. Cell death was decreased but not prevented by continued presence of 10 mmol/l nicotinamide and apoptosis decreased by 24 h incubation with 20 micromol/l Z-VAD. Insulin secretion was maintained over 6 days following treatment with both agents.


CONCLUSIONS
Purification of human beta cells induces marked apoptosis limiting their function and survival in vitro. This was improved by matching the extracellular matrix to the specific expression of integrins and by addition of nicotinamide and Z-VAD.
Ris
F
F
Louis-Jeantet Research Laboratories, University of Geneva Medical Center and Division of Surgical Research, Department of Surgery, University Hospital, Geneva, Switzerland.
Hammar
E
E
Bosco
D
D
Pilloud
C
C
Maedler
K
K
Donath
M Y
MY
Oberholzer
J
J
Zeender
E
E
Morel
P
P
Rouiller
D
D
Halban
P A
PA
D000328:Adult; D000368:Aged; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D061945:Caspase Inhibitors; D002469:Cell Separation / Q000379:methods; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D005109:Extracellular Matrix / Q000502:physiology*; D005347:Fibroblasts / Q000166:cytology; D005434:Flow Cytometry; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D007328:Insulin / Q000235:genetics; D016023:Integrins / Q000235:genetics* / Q000502:physiology*; D007515:Islets of Langerhans / Q000166:cytology*; D008164:Luminescent Proteins / Q000235:genetics; D008875:Middle Aged; D009536:Niacinamide / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis; D014162:Transfection
D061945:Caspase Inhibitors; D017077:Culture Media, Conditioned; D007328:Insulin; D016023:Integrins; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D009536:Niacinamide