Proliferation of sorted human and rat beta cells
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The aim of the study was to determine whether purified beta cells can replicate in vitro and whether this is enhanced by extracellular matrix (ECM) and growth factors.
Human beta cells were purified by FACS by virtue of their high zinc content using Newport Green, and excluding ductal and dead cells. Rat beta cells were sorted by autofluorescence or using the same method developed for human cells. Cells were plated on poly-l-lysine or ECMs from rat or human bladder carcinoma cells or bovine corneal ECM and incubated in the presence of BrdU with or without growth factors.
The newly developed method for sorting human beta cells yields a population containing 91.4 ± 2.8% insulin-positive cells with a low level of spontaneous apoptosis and a robust secretory response to glucose. Beta cells from 8-week-old rats proliferated in culture and this was increased by ECM. Among growth factors, only human growth hormone (hGH) and the glucagon-like peptide-1 analogue liraglutide enhanced proliferation of rat beta cells, with a significant increase on both poly-l-lysine and ECM. By contrast, sorted adult human beta cells from 16 donors aged 48.9 ± 14.3 years (range 16–64 years) failed to replicate demonstrably in vitro regardless of the substratum or growth factors used.
These findings indicate that, in our conditions, the fully differentiated human adult insulin-producing beta cell was unable to proliferate in vitro. This has important implications for any attempt to expand cells from pancreases of donors of this age group. By contrast, the rat beta cells used here were able to divide in vitro, and this was enhanced by ECM, hGH and liraglutide.
KeywordsBeta cells Extracellular matrix Growth factors Insulin secretion Pancreatic islets Proliferation
bovine corneal endothelial extracellular matrix
carbohydrate antigen 19-9
glucose-stimulated insulin secretion
human growth hormone
Juvenile Diabetes Research Foundation
Krebs–Ringer bicarbonate HEPES buffer
pancreatic and duodenal homeobox 1
As the availability of human donors for pancreatic islet transplantation is limited, there is an urgent need for alternative sources. In vitro expansion of adult human beta cells may provide an adequate source for beta cell therapy, but little is known about their proliferative capacity.
The extracellular matrix (ECM) is a complex structural entity surrounding cells within mammalian tissues that is able to regulate many essential cellular functions, including proliferation [1, 2, 3, 4]. ECM produced by 804G (rat bladder carcinoma) cells thus improves glucose-stimulated insulin secretion (GSIS) and survival of primary rat beta cells [5, 6, 7]. Furthermore, this ECM or its human equivalent (HTB9-ECM) increases replication of human endocrine islet and/or duct cells [8, 9, 10, 11].
Several growth factors have been shown to be mitogenic for insulinoma cell lines [12, 13, 14, 15, 16], but their ability to promote replication of adult beta cells has been less extensively studied. The focus of this study was to understand whether adult beta cells can replicate in vitro, and if so whether this can be enhanced by ECM and growth factors.
Rat islet isolation and beta cell purification
Animal experimentation conformed to the guide for the care and use of laboratory animals and was authorised by the Veterinarian Office of Canton de Geneve (Geneva, Switzerland). Islets were isolated by collagenase digestion of pancreases from 2- or 18-month-old male Wistar rats (Janvier, Le Genest-St-Isle, France), followed by Ficoll purification as described . Islets were trypsinised and beta cells purified using a FACStar-Plus (Becton Dickinson, Sunnyvale, CA, USA) as described [18, 19] by autofluorescence or by virtue of their zinc content using the zinc-binding fluorochrome Newport Green (NG).
Human beta cell purification
Human islets (average age of cadaveric donors 48.9 ± 14.3 years; range 16–64 years; n = 16) were provided through the Juvenile Diabetes Research Foundation (JDRF) Islet Distribution Program by Islet Cell Resource Centers in Geneva (Switzerland), Milan (Italy) and Lille (France). The use of human tissue for research was approved by our local institutional ethical committee.
Single cell suspensions were obtained by incubating (37°C, 10 min) islets in Accutase (PAA Laboratories GmbH, Colbe, Germany). The dispersed cells were washed with modified Krebs–Ringer bicarbonate HEPES buffer (KRBH: 125 mmol/l NaCl, 4.74 mmol/l KCl, 1 mmol/l CaCl2, 1.2 mmol/l KH2PO4, 1.2 mmol/l MgSO4, 5 mmol/l NaHCO3, 25 mmol/l HEPES, pH 7.4, 0.5% BSA) containing 2.8 mmol/l glucose and resuspended in this buffer. Cells were incubated (1 h, 4°C) with mouse anti-pan-ductal membrane carbohydrate antigen 19-9 (CA19-9) antibody (1 μg/ml; NovoCastra, Newcastle upon Tyne, UK), rinsed twice with KRBH and reincubated for 1 h with Alexa-Fluor 633 anti-mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing, cells were resuspended in KRBH containing 1.5 μl/ml Pluronic F127 and incubated (30 min, 37°C) with 1 μmol/l NG-PDX-Ac (Molecular Probes, Leiden, The Netherlands). After washing, cells were stained 10 min with 7-amino-actinomycin-D (7-AAD, 10 μg/ml; Molecular Probes). Analysis and cell sorting were performed using a FACSvantage (Becton Dickinson). Beta cells were enriched in the 7-AAD-negative, CA19-9-negative and NG-bright populations.
ECM from 804G cell-conditioned medium was prepared and used as described previously . For lawns of lysed cells, HTB9 (human bladder carcinoma) cells were grown to confluency and then lysed by exposure for 10 min to 0.1 mol/l NH4OH followed by washes with H2O (adapted from ). Coated Petri dishes were stored at 4°C after air-drying. Before use, dishes were washed once again and dried. Bovine corneal ECM (BC-ECM)-coated plasticwares were from Biological Industries (Kibbutz Beit Haemek, Israel). Dishes coated with poly-l-lysine (PLL; 100 μg/ml) were used as control.
Culture of cells
Culture medium for rat beta cells was DMEM (Gibco, Invitrogen, Basel, Switzerland), 10% FCS, 11.2 mmol/l glucose, 1 mmol/l sodium pyruvate, 110 U/ml penicillin, 110 μg/ml streptomycin and 50 μg/ml gentamicin. For human beta cells, medium was CMRL 1066, 10% FCS, 1% glutamine, 5.6 mmol/l glucose, 1 mmol/l HEPES and antibiotics as above. Sorted cells were washed with appropriate medium and incubated (20 h, 37°C) in non-adherent 100 mm diameter Petri dishes to allow recovery from cell purification. Cells were resuspended at 4 × 105 cells/ml in medium, and 50 μl droplets deposited on coated dishes. Intact human islets were similarly deposited as droplets (ten islets per dish). In each instance, after 24 h, 2 ml culture medium were added to the droplets.
Sorted cells were attached (90 min) to PLL-coated Cunningham’s chambers and then fixed (4% paraformaldehyde [wt/vol.], 20 min). After washing (PBS), cells were incubated (PBS/0.5% Triton X-100 [wt/vol.], 4 min), rinsed (PBS), preincubated (30 min, room temperature) in PBS/0.5% BSA (wt/vol.) and exposed for 2 h (room temperature) to guinea pig anti-insulin serum (DB, University of Geneva, Switzerland) and rabbit anti-glucagon antibody (Dako, Baar, Switzerland). After three washes they were incubated (1 h, room temperature) with FITC-conjugated goat anti-rabbit or rhodamine-conjugated goat anti-guinea pig antibodies diluted in PBS/0.5% BSA (wt/vol.). Samples were examined by fluorescence microscopy (Carl Zeiss, Feldbach, Switzerland).
Beta cells were washed three times with KRBH, 2.8 mmol/l glucose and preincubated (1 h, 37°C) with this same buffer, followed by successive 1 h incubations at 37°C in KRBH with 2.8 and 16.7 mmol/l glucose for rat beta cells or 22.2 mmol/l glucose for human beta cells. When indicated, growth factors were added directly in KRBH containing 16.7 mmol/l glucose. Cellular insulin was extracted with acid–ethanol, and insulin in buffers and extracts measured by RIA with rat or human insulin as the standard. Total insulin is the sum of extracted insulin and insulin secreted during the two incubations.
Detection of proliferation by immunofluorescence
Islets or sorted beta cells were incubated with 10 μmol/l BrdU for 1–6 days. They were fixed (1% paraformaldehyde [wt/vol.], 1 h room temperature) and DNA denatured (1.5 mol/l HCl). After permeabilisation (PBS/0.5% Triton X-100 [wt/vol.], 4 min), proliferation was estimated using an immunohistochemical assay kit as described by the manufacturer (BrdU Labelling and Detection Kit; Roche, Basel, Switzerland). For Ki-67 staining, cells were fixed after 2–9 days of culture, washed (PBS), permeabilised (PBS/0.1% Triton X-100 [wt/vol.], 10 min), rinsed (PBS), preincubated (30 min, room temperature) in PBS/1% BSA (wt/vol.) and stained with mouse anti-Ki-67 (BD Pharmingen, Basel, Switzerland) for 2 h (room temperature), followed by Alexa-fluor 533-conjugated anti-mouse Ig. Cells were also co-stained for insulin or for pancreatic and duodenal homeobox 1 (PDX1; goat anti-PDX1; kind gift from C. V. Wright, Vanderbilt University, Nashville, TN, USA). Following washing, cells were incubated with Alexa-fluor 488-conjugated anti-guinea pig or anti-goat Ig. Nuclei were stained with 10 μg/ml DAPI (Sigma-Aldrich, Buchs, Switzerland). The percentage of cells double-positive for insulin+BrdU or PDX1+BrdU per total number of insulin- or PDX1-single-positive cells, respectively, was calculated.
To study effects of exogenous growth factors, beta cells were cultured for 1–5 days with 40 nmol/l of the long-acting glucagon-like peptide-1 (GLP-1) analogue liraglutide, 0.5 μg/ml EGF, 2 μmol/l gastrin, 400 ng/ml human growth hormone (hGH) (the generous gift of Novo Nordisk, Bagsvaerd, Denmark; concentrations were as recommended by the supplier and in the range of concentrations used by others ) and 2 nmol/l betacellulin (Sigma Buchs; a concentration that had been shown to increase DNA synthesis in human fetal islet-like cell clusters ).
Data are means±SEM for n independent experiments (using cells from separate islet isolations) unless specified otherwise. Statistical significance was tested by Student’s unpaired t test with p < 0.05 considered significant.
Effect of ECM on rat beta cell proliferation
Purity of human beta cell preparations
Since it proved impossible to obtain a suitably enriched population of human beta cells using the same (autofluorescence) method as for rat cells, we adapted and combined previously published techniques [23, 24, 25] for this purpose, neither of which proved satisfactory alone. After dissociation of islets, human ductal cells were specifically labelled using anti-CA19-9 antibody, beta cells were labelled with NG (due to their high zinc content) and dead cells were labelled with 7-AAD, which binds to DNA when cell membrane permeability is altered after cell death. Cells were then analysed by FACS.
To determine whether the NG sorting method could impact beta cell proliferation, rat beta cells sorted by this method were compared with those sorted by autofluorescence. Proliferation was similar and 804G-ECM significantly increased cell proliferation in both instances (Electronic supplementary material Fig. 1).
Effect of ECM on human beta cell proliferation
To investigate the hypothesis that human beta cells can lose insulin labelling during replication (perhaps by degranulation), we labelled beta cells for PDX1, a transcription factor expressed by beta cells in adult islets. After 4 days in the presence of BrdU, we failed to observe any colocalisation between BrdU and PDX1 (Fig. 5d) either in >500,000 sorted beta cells or in approximately 200 intact islets obtained from four islet preparations that were evaluated in this regard.
With increasing culture time we observed the appearance of fibroblastoid- or mesenchymal-like cells with a large oval nucleus in sorted beta cells and in (unsorted) dispersed islet cell populations. Most of these cells were BrdU-positive (Fig. 5d) and were far more abundant in dispersed islet cells. These cells were systematically negative for insulin or PDX1.
Effect of growth factors on beta cell proliferation and insulin secretion
Since human beta cell replication could not be stimulated by the growth factors, it was possible that they were unresponsive in all respects to these agents. To investigate this, we tested their effect on insulin secretion. As expected, liraglutide significantly increased insulin secretion from both rat and human beta cells plated either on PLL or on the different matrices (Fig. 7c,d). For rat beta cells, hGH had a minor but significant effect on GSIS both on cells plated on PLL and on 804G-ECM (Fig. 7c).
Effect of age on rat beta cell proliferation
To investigate the hypothesis that age can influence the proliferative capacity of beta cells, we also analysed beta cell proliferation from 18-month-old rats. Beta cells obtained from 18-month-old rats proliferated in culture and 804G-ECM increased this proliferation (0.84 ± 0.37% BrdU-positive beta cells on PLL vs 2.87 ± 0.1% on 804G-ECM; 24 h incubation with BrdU; mean±SD, n = 2 independent experiments). However, in this limited series of experiments, beta cells from these old rats did appear to proliferate less than those from young (8-week-old) rats both on PLL and on 804G-ECM (1.96 ± 0.01% and 4.85 ± 0.97%, respectively).
In this study we describe a new technique using NG staining and autofluorescence-activated flow cytometry to obtain a >90% pure and functional population of beta cells. Using this and an established method for sorting rat beta cells, we studied the effect of ECM in combination with growth factors on beta cell proliferation. In previous studies intact islets or islet cells more or less spread on ECM were used to examine beta cell replication. In these conditions, overlay of cells can lead to ambiguous labelling. Indeed the report that human beta cells are able to replicate when exposed to selected matrices and growth factors  was challenged by others showing that these culture conditions were mitogenic for duct cells and not for beta cells . Using sorted human beta cells, and regardless of the culture conditions (various ECMs with or without growth factors), we failed to observe, according to our criteria, any bona fide beta cells that are Ki-67-positive or that had incorporated BrdU even during prolonged (>10 days) culture. We cannot exclude that an even longer time could be required for proliferation to be induced. However, and in striking contrast, beta cells from young rats were seen to proliferate in vitro and this was significantly increased by ECM and growth factors. The technique used to purify human beta cells is not the cause, because rat beta cells, purified with the same NG sorting method used for human cells, can proliferate in a similar manner to when sorted using the well-established autofluorescence method. Control experiments further indicated that neither the ECM nor the medium used for human beta cells was the cause. Furthermore, it was not possible to detect beta cell proliferation even in (intact) isolated human islets. To exclude the fact that division of human beta cells could be accompanied by rapid loss of insulin, we also performed double immunostaining of BrdU or Ki-67 and PDX1. But again no colocalisation of BrdU or Ki-67 with PDX1 was observed in human beta cells, in contrast to results obtained using rat beta cells.
The divergence between our data and those of Beattie et al. [8, 9, 10] may be explained in part, by differences in methods. In their studies, proliferation was on some occasions quantified by 3H-labelled thymidine incorporation, which determines the proliferation of all cells and not only beta cells. However, on other occasions proliferating cells were double-stained for insulin and BrdU  or Ki-67 . As discussed above, however, even with confocal microscopy it is not easy to determine whether cells are truly double-labelled or not if they are not in a veritable monolayer. Beattie et al. conclude that the cells they identify as double-positive are never ‘brightly stained’ for insulin [9, 10], whereas upon close inspection of many such cells we consider them to be insulin-negative. In our study we did observe, as others in the past, a growing population of cells that were BrdU- or Ki-67-positive, but it was apparent by confocal microscopy that all of these cells were insulin or PDX1-negative. Moreover these cells that closely resemble the human islet-derived mesenchymal-like cells described by others [27, 28] were more abundant when we cultured dispersed islet cells rather than purified beta cells, suggesting that they did not derive from beta cells by epithelial–mesenchymal transition . This supports recent studies showing that mesenchymal cells emerging from mouse islets are similarly not derived from beta cells [29, 30, 31, 32]. Nevertheless, we cannot exclude from our studies that in human beta cells proliferation is accompanied by the rapid loss of characteristic features such as PDX1 and insulin expression or that their expression is sufficiently attenuated as to make it undetectable in our immunofluorescence conditions. This issue could only be resolved by labelling human beta cells with a stable marker that is not lost upon de-differentiation. Double-positive cells (insulin and ductal markers) have been observed in healthy and pathological human pancreases . These cells may represent newly differentiated ductal precursor cells as well as beta cells which have de-differentiated towards a ductal precursor. Our technique for sorting beta cells would have excluded such cells, but had such cells proliferated in the unsorted islet cells or whole islet preparations they would have been scored as ‘insulin-positive’ cells, regardless of whether they also expressed ductal markers: no such cells were detected. However, interestingly, lineage tracing in mouse beta cells shows that de-differentiation occurs but is not followed by proliferation . Furthermore, and although cells from 16 different human pancreases were studied, with thousands of cells screened in each preparation, we cannot exclude that adult human beta cells can replicate under our conditions at a non-detectably low rate or that other conditions may exist that might allow for such proliferation.
ECM has been reported to enhance proliferation of other cell types in vitro , but this is the first report to show that primary rat beta cell proliferation is significantly increased by ECM. The signal transduction mechanism involved in this effect remains to be determined, but integrins could play a role in this phenomenon as has been demonstrated for insulin secretion .
Among the growth factors and hormones tested, only hGH and the GLP-1 analogue significantly increased adult rat beta cell proliferation. hGH levels rise during pregnancy and lactation, states that are associated with an expansion of the beta cell mass [36, 37], and hGH is one of the most potent mitogenic stimuli for rat beta cells even if its effect is greater in neonatal than in adult rat [38, 39]. hGH was shown many years ago to stimulate human islet cell proliferation . However, direct comparison with the present study is not possible for several reasons. First, incorporation of BrdU into all islet cells was quantified in the older study, without identification of cell type. Furthermore, only two human islet preparations were studied for their proliferation and hGH only increased proliferation in one. Although it is intriguing to note that these hGH-responsive islets were from a relatively young (16-year-old) female donor pancreas, while the other was from a 49-year-old female, it is impossible to draw any conclusions from such a limited sample set.
In our study, the GLP-1 analogue liraglutide significantly increased proliferation of rat beta cells only after 48 h but not 24 h of treatment, but instantly increased GSIS, in keeping with its well known insulinotrophic action [40, 41, 42, 43]. Several in vitro studies have also shown that GLP-1 and its analogues are capable of inducing beta cell proliferation in rodent islets and insulinoma cell lines [14, 21, 41]. The proliferative effects of GLP-1 appear to involve multiple cellular pathways which may involve transcriptional induction of cyclin D1 . Furthermore, GLP-1 also induces Pdx1 gene expression . Taken together, GLP-1 could induce gene expression necessary for cell division, possibly explaining the need for a 48 h exposure to the hormone.
The effect of ECM on proliferation seemed to be additive to that of either hGH or liraglutide, suggesting an independent mode of action. In this study, betacellulin did not increase rat or human beta cell replication even though this member of the EGF family has been identified as a beta cell mitogen in INS-1 cells . But in fact, in the rat RINm5F cell line, and also in fetal human beta cells, betacellulin failed to induce proliferation [16, 22], suggesting that the type and stage of development of the cells used can influence the outcome.
The discrepancy between the results obtained in rat and human beta cells, apart from an intrinsic species difference, could be explained by the age of the pancreas used for islet isolation (for most experiments, rat beta cells were obtained from 8-week-old animals whereas human beta cells were obtained from patients with an average age of 49 years). This hypothesis is supported by recent work from others demonstrating slower beta cell turnover in aged mice  or rats , and is favoured by our own results suggesting a lower proliferative capacity in beta cells isolated from 18-month-old rats even though the limited number of experiments does not allow for statistical analysis. Nevertheless, the old rat beta cells did proliferate in vitro in marked contrast to human beta cells (from all donors in the present study, spanning the range 16–64 years of age). Therefore, it seems that the observed discrepancy between rat and human beta cells probably reflects the combination of species as well as age differences. Regardless, the purpose of this study was not to compare beta cell proliferation from age-matched rat and human pancreases, but rather to evaluate the proliferative capacity of fully differentiated human beta cells from donors within the typical age range used for transplantation or experimental purposes. Rat beta cells were used as a positive control. Our (negative) results using human beta cells in our experimental setting have important implications for any attempt to expand cells from pancreases of donors of this age group (16–64 years).
In summary, rat beta cells can proliferate in vitro. This is significantly increased by ECM with a further additive increase by hGH and a GLP-1 analogue. Indeed and strikingly, after 6 days of continuous culture with BrdU, more than half of the cells on 804G-ECM had become labelled with BrdU (60.44 ± 3.08%, n = 3), an extraordinarily high number for a fully differentiated cell type long considered essentially quiescent. By contrast, in this study using islets from cadaveric organ donors above the age of 16 years, we failed to detect replication of the fully differentiated human adult beta cell in vitro regardless of whether they had been sorted by FACS or not, and of the culture environment. While these negative data do not allow us to draw any conclusions regarding in situ beta cell regeneration in vivo in adult humans, and the possible beneficial effects of factors such as GLP-1 in such a context, they do have important implications for any attempt to grow such cells in culture with a view to obtaining increased numbers for transplantation.
This work was supported by the JDRF Program for Regeneration of Beta Cell Function (Grant 1-2005-826). Human islets of Langerhans were provided by the Cell Isolation and Transplantation Center at the University of Geneva School of Medicine, Switzerland, by the Department of Surgery, INSERM U859, Lille 2 University, Lille, France, and by the Islet Processing Facility, S. Raffaele Scientific Institute, Milan, Italy, thanks to the European Consortium for Islet Transplantation ‘Islets for Research’ Distribution Program sponsored by the JDRF. We thank C. Raveraud-Rouget for expert technical assistance, Novo Nordisk A/S for research support to T. Mandrup-Poulsen, N. Billestrup and C. Bruun, and A. E. Karlsen for the generous gift of the growth factors and hormones used in this study.
Duality of interest
G. Parnaud, D. Bosco, T. Berney, F. Pattou, J. Kerr-Conte, M. Y. Donath, C. Bruun, N. Billestrup and P. Halban declare that there is no duality of interest associated with this manuscript. T. Mandrup-Poulsen is employed by Novo Nordisk and is also a shareholder and has received grants from Novo Nordisk.