RAPID COMMUNICATION |
Assessment of Human Pancreatic Islet Architecture and Composition by Laser Scanning Confocal Microscopy
Department of Medicine, Division of Diabetes, Endocrinology, and Metabolism, Vanderbilt University School of Medicine, Nashville, Tennessee (MB,MJF,WEN,AC,ACP); Islet and Autoimmunity Branch of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland (BH,DMH); and VA Tennessee Valley Healthcare System, Nashville, Tennessee (ACP)
Correspondence to: Alvin C. Powers, Division of Diabetes, Endocrinology, and Metabolism, 715 PRB, Vanderbilt University, Nashville, TN 37232. E-mail: Al.Powers{at}Vanderbilt.edu
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Summary |
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(J Histochem Cytochem 53:10871097, 2005)
Key Words: pancreatic islets confocal microscopy architecture composition
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Introduction |
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Materials and Methods |
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Mouse Islet Isolation
Islets from mice of three different strains were isolated by dissection of the splenic portion of pancreas followed by digestion with collagenase P (Boehringer Mannheim; Indianapolis, IN) (Brissova et al. 2002). Groups of two pancreata were digested in 2 mg collagenase/pancreas in Hanks' balanced salt solution (0.6 mg collagenase/ml) for 69 min at 37C using a wrist-action shaker. Mouse islets were subjected to immunohistochemical analysis immediately after isolation.
Human and Non-human Primate Islets
Human islets were prepared by the Cell Processing Unit, Department of Transfusion Medicine, National Institutes of Health (Hirshberg et al. 2003a) or obtained through the Juvenile Diabetes Research Foundation Human Islet Distribution Program [islets studied were provided by the Diabetes Institute for Immunology and Transplantation at the University of Minnesota (Bernhard J. Hering, Jeffrey Ansite, and Hui-Jian Zhang) and The Diabetes Research Institute Islet Cell Resource at the University of Miami (Camilo Ricordi)]. Non-human primate islets from rhesus macaques were prepared by the Islet and Autoimmunity Branch of the NIH (Hirshberg et al. 2002a
,b
). After isolation, human and non-human primate islets were shipped in CMRL media by overnight courier to Vanderbilt University and cultured for an additional 24 hr in CMRL media, 95% CO2/5% O2 at 37C. Human and non-human primate islets were subjected to immunohistochemical analysis
48 hr after isolation (approximate time required for islet shipping). At the same time, the health of human islet preparations was examined in the cell perifusion system (Wang et al. 1997
) and perifusate fractions were assayed for insulin by radioimmunoassay (Brissova et al. 2002
). Four hundred-islet equivalents were perifused at 1 ml/min flow rate and 3-min fractions were collected after a 30-min equilibration period in 2.8 mM glucose. In addition to 16.8 mM glucose, two islet preparations were also tested for their responsiveness to a combination of 16.8 mM glucose + 250 µM IBMX and 2.8 mM glucose + 300 µM tolbutamide.
Antibodies
Guinea pig anti-human insulin IgG (1:1000) and rabbit anti-glucagon IgG (1:5000) were from Linco Research, Inc. (St. Charles, MO), sheep anti-somatostatin IgG (1:1000) was from American Research Products, Inc. (Belmont, MA). The antigens were visualized using appropriate secondary antibodies conjugated with Cy2, Cy3, and Cy5 fluorophores (1:1000) from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA).
Immunocytochemistry Performed on Histological Sections
Dissected adult mouse pancreata were rinsed in ice-cold 10 mM PBS and fixed in freshly prepared 4% paraformaldehyde (Electron Microscopy Sciences; Washington, PA)/100 mM PBS for 1.5 hr on ice. Following fixation, the tissues were washed four to six times with 100 mM PBS over a period of 2 hr and then equilibrated in 30% sucrose/10 mM PBS overnight at 4C. The tissues were cryopreserved in optimum cutting temperature compound (VWR Scientific Products; Willard, OH) at 80C, and 8-µm sections were mounted on charged slides. Human pancreatic tissue obtained from the NIH (Hirshberg et al. 2003a) was processed the same way as the mouse pancreas. Cryosections were permeabilized in 0.2% Triton X-100 for 10 min at room temperature, blocked with 5% normal donkey serum (Jackson ImmunoResearch Laboratories, Inc.) for 1.5 hr, and then incubated with primary antibodies overnight at 4C. Secondary antibodies were applied to the tissue sections for 1 hr at room temperature. Both primary and secondary antibodies were diluted in 10 mM PBS containing 1% BSA and 0.1% Triton X-100. Digital images of the 8-µm cryosections mounted with AquaPoly/Mount (Polysciences; Warrington, PA) were acquired with a MagnaFire digital camera (Optronics; Goleta, CA) connected to an Olympus BX-41 fluorescence microscope (Olympus; Tokyo, Japan).
Immunocytochemistry Performed on Wholemount Islets
Freshly isolated islets were attached to MatTek dishes (Cat. #P35G-0-14-C; MatTek Corporation, Ashland, MA) precoated with CELL-TAK adhesive (Becton Dickinson Labware; Bedford, MA) and fixed in 4% paraformaldehyde/10 mM PBS for 25 min at room temperature. To ensure that the entire islet was optically sectioned, we chose islets ranging from 60 µm to 100 µm in (z) dimension for study. Because islets are known for not having a perfectly spherical shape, their size in (x) and (y) dimensions varied as much as from 60 µm to 200 µm. The fixation was followed by three 30-min washes in 10 mM PBS and 3-hr permeabilization with 0.3% Triton-X 100/10 mM PBS. The islets were blocked with 5% normal donkey serum/0.15% Triton-X 100/10 mM PBS overnight at 4C and then equilibrated in antibody dilution buffer twice for 20 min at room temperature. The primary and secondary antibodies were diluted in 1% BSA/0.2% Triton X-100/10 mM PBS and the incubations were carried out for 24 hr at 4C. The islets were mounted with AquaPoly/Mount (Polysciences). Samples were subjected to optical sectioning at 1-µm increments in axial (z) dimension using a Zeiss LSM410 confocal laser scanning microscope (Carl Zeiss; Jena, Germany).
Quantification of Endocrine Cell Types in the Isolated Islets
Optical sections of isolated islets were analyzed and three-dimensionally (3-D) reconstructed using MetaMorph v6.1 software (Universal Imaging Corporation; Downington, PA). Calibrated RGB overlays of the z-stacks for each individual islet were color thresholded, and then the contribution of islet cells labeled in red (VR, cells), green (VG, ß cells), and blue (VB,
cells) to the islet volume (V = VR + VG + VB; µm3) was measured. The population of each islet cell type was expressed as a fraction of the total islet volume: VR/V; VG/V; VB/V (%).
Statistical Analysis
Unpaired t-test was used to compare populations of ß, , and
cells in mouse and human islets.
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Results |
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Discussion |
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Histological sections of the human pancreas, however, have a different appearance than those of the mouse or rat. Micrographs of human islets published by Orci and colleagues (1976), who at that time studied changes in islet morphology associated with type 1 diabetes, suggest that
and
cells in the adult human islets are dispersed throughout the islet rather than being confined to its periphery. More recent data from the study by Dubois and colleagues (2000)
, who were interested in PPAR
expression in normal human pancreatic islets, also suggest that
and
cells in adult human islets are arranged in a less stereotypical fashion than in mouse islets. Although histological sections of the human pancreas suggest a distinct architecture for human islets compared with rodent islets, without a serial sectioning through an entire islet, one cannot be certain whether the sections were collected from the mantle (more intermingling of ß and non-ß cells) or a deeper portion of the islet comprising the core. Using confocal laser scanning microscopy, Brelje and colleagues (1989)
previously described the presence of
,
, and PP cells deeper within the isolated human islets. However, their study was limited by the depth to which they were able to section into the specimens. By acquisition of serial optical sections through the entire isolated human islets, we were able to demonstrate that human islets lack the typical coremantle architecture of mouse/rat islets and that ß cells are rather intermingled with
and
cells. Furthermore, ß cells are commonly on the surface of isolated human islets. This observation was consistent across six different human islet preparations (from three different human islet isolation centers) and islets of various sizes. Interestingly, non-human primate islets displayed architecture similar to human islets.
Our data indicate that human islets not only have an architecture distinct from that of rodent islets, but that endocrine cell populations are also quite different in human islets. In the human pancreas, endocrine cell populations were previously examined with respect to their location in the gland (Orci et al. 1976; Gersell et al. 1979
; Malaisse-Lagae et al. 1979
), but it is not known what populations of endocrine cell types are present in individual islets. For example, Malaisse-Lagae and colleagues (1979)
, who identified a PP-rich lobe in the adult human pancreas, noticed a rather significant variation in ß- and
-cell populations between PP-rich and PP-poor pancreatic regions. An independent study by Gersell and colleagues (1979)
confirmed partition of the human pancreas into PP-rich and PP-poor pancreatic regions by radioimmunoassay and in addition found that a similar phenomenon exists in the canine pancreas. In our studies we selected islets isolated from the total pancreas; therefore, we cannot ascertain the original islet location. However, because we examined a number of islets randomly selected from different islet preparations isolated at three different isolation centers, it seems unlikely that our islets reflect an ascertainment bias. It is highly unlikely that out of an estimated 6,000,000 human islets we would have randomly picked 32 islets that were significantly architecturally and compositionally different from 28 randomly picked mouse islets (three different isolations, 200 islets/isolation). Furthermore, the architecture of intact islet correlated with histological sections of human pancreas. Thus, we feel that our results are representative of islets isolated for human islet transplantation; this concept is supported by the observation that similar islet cell populations were seen in dispersed islets used for human islet transplantation (Shapiro et al. 2000
). In addition, from work by Orci and colleagues, it appears that endocrine cell populations in the human pancreas may change throughout life (Malaisse-Lagae et al. 1979
; Orci et al. 1979
). In infant pancreas, they reported a higher abundance of
cells, which happens at the expense of PP cells in the PP-rich region and ß cells in the PP-poor region. This has not been examined in detail in the pancreas of other species.
Similar heterogeneity of endocrine cell types was observed when Redecker and colleagues (1992) studied the microanatomy of canine islets and used semi-thin sections (up to 1000 sections/islet) to reconstruct the islets. A large number of serial histological sections (up to 77 sections/islet) were also used for the reconstruction of individual rat islets and the examination of their endocrine cell populations (Beatens et al. 1979
). These investigators found that PP-rich islets were located in the ventral pancreas, whereas islets in the dorsal pancreas had a lower abundance of PP cells. However, the population of ß cells in the PP-rich and PP-poor rat islets was remarkably similar (82.5 vs 82.0%), and only the
-cell population varied. The homogeneous ß-cell distribution in the rat islets contrasts with the fluctuation of ß and
cells that we found within individual human islets and that was previously described in PP-rich and PP-poor regions of the human pancreas. The murine islets examined in our study were very similar to the observations with rat islets (Beatens et al. 1979
), not only by their architecture but also in terms of uniformity in the cellular composition. Thus, these results suggest that islet composition in higher mammals (human, non-human primate, and canine) is more diverse than in rodent islets.
Our findings have several theoretical and practical implications: (a) factors that control human islet development and structure may differ from those in the mouse and thus we encourage caution in extrapolating findings in rodent models directly to higher species including man; (b) beta cells in isolated human islets are often on the islet surface and thus may be more accessible than rodent beta cells (for example, for gene transfer approaches); and (c) human islet heterogeneity may impact assessments of islet quality for transplantation and determinations of insulin secretory capacity.
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Acknowledgments |
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We thank Drs. Camillo Ricordi [The Diabetes Research Institute Islet Cell Resource (ICR) at the University of Miami] and Bernhard Hering (Diabetes Institute for Immunology and Transplantation at the University of Minnesota) for assistance in providing human islets for study.
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Footnotes |
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Literature Cited |
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