Identification and Purification of Functional Human β-cells by a New Specific Zinc-fluorescent Probe

Abstract

S U M M A R Y Pancreatic ␤ -cells contain large amounts of zinc. We took advantage of this to try to localize, quantify, and isolate insulin-producing cells from islet preparations. Our study was designed to identify a non-toxic zinc-sensitive fluorescent probe able to selectively label labile zinc in viable ␤ -cells and to exhibit excitation and emission wavelengths in the visible spectrum, making this technique exploitable by most instruments. We tested Newport Green, a probe excitable at 485 nm with a dissociation constant in the micromolar range corresponding to a low affinity for zinc. The loading of the lipophilic esterified form of Newport Green was easy, rapid, specific, and non-toxic to cells. Confocal microscopy highlighted an intense fluorescence associated with secretory granules. Regression analyses showed a good relationship between zinc fluorescence and islet number ( r ϭ 0.98) and between zinc fluorescence and insulin content ( r ϭ 0.81). The determination of Zn fluorescence per DNA enabled us to assess the quality of the different islet preparations intended for islet allografting in terms of both purity and viability. Cell sorting of dissociated Newport Green-labeled cells resulted in a clear separation of ␤ -cells, as judged by insulin content per DNA and immunocytochemical analysis. This zinc probe, the first able to specifically label living cells in the visible spectrum, appears very promising for ␤ -cell experimentation, both clinically and for basic research. P ancreatic islets contain a much higher concentration of zinc than other tissues. Zinc plays an important role in packaging insulin because it is firmly established as an integral part of the insulin crystal as a 2-Zn-insulin hexamer. In addition, free ionized zinc is found in the extragranular space of ␤ -cells, where it may act as a reservoir for granular zinc (Figlewicz et al. 1984). These pools of free and loosely bound zinc can be visualized histochemically by most cytological stains, unlike zinc tightly complexed to proteins such as metalloproteins, including transcription factors and metalloenzymes. Our study was designed to search for a new non-toxic probe that was able to specifically stain living ␤ -cells. Our aim was threefold: (a) to visualize ␤ -cells in pancreatic islets by confocal microscopy or image analysis; (b) to isolate these ␤ -cells for physiological studies; and (c) to rapidly estimate, in allograft management, the amount of viable ␤ -cells in semipurified preparations containing contaminating exocrine cells. The latter pretransplantation evaluation of the quality of the graft has become a crucial prerequisite to ensure successful engraftment. Here we describe the use of Newport Green, a new zinc-sensitive probe with an excitation wavelength in the visible spectrum, which fulfills all the criteria required for efficient staining of human ␤ -cells in clinical and research applications. Materials and Methods Human Islet ProcessingHuman pancreata ( n ϭ 11; mean age 35 Ϯ 12 years) were harvested from adult brain-...

Inhibition of c-jun N terminal kinase (JNK) improves functional beta cell mass in human islets and leads to AKT and glycogen synthase kinase-3 (GSK-3) phosphorylation

“…Interestingly, recent data suggest that JNK may represent a key target factor of such an inflammatory response [1,33,34], since activation of JNK during islet isolation and engraftment is detrimental to islet cell survival [4,5,9,24]. Both ATP-competitive and noncompetitive JNK inhibitors have shown biological activity in pancreatic islets [4,9], although we and others have shown that some cell-permeable peptides may be toxic to islet cells [5,8,12].…”

“…Measurement of viable beta cells was performed as described [23]. Briefly, the zinc-binding dye Newport green (NG) allows identification and quantification of islet beta cells on dissociated islets, based on the abundant zinc content in beta cell secretory granules [24]. The membrane exclusion dye 7-aminoactinomycin-D (7AAD; Invitrogen, Carlsbad, CA, USA) was used as marker of cell death.…”

Identification of Insulin‐Producing Cells Derived from Embryonic Stem Cells by Zinc‐Chelating Dithizone

“…DTZ produces fluorescence when dissolved in DMSO, but its fluorescence fades too quickly for reliable sorting [44]. Recently, a new specific zinc-fluorescent probe, Newport Green, has been reported to be easy, rapid, specific, and nontoxic to insulin-producing cells [45]. Instead of DTZ, the use of Newport Green may be helpful for the purification and collection of insulin-producing cells from in vitro differentiated ES derivatives by cell sorting.…”

Impact of integrin-matrix matching and inhibition of apoptosis on the survival of purified human beta-cells in vitro

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