First published online May 11, 2005
Stem Cells
Vol. 23 No.
7
August 2005, pp.
862
-867
doi:10.1634/stemcells.2004-0288; www.StemCells.com
© 2005 AlphaMed Press
Sequestration and Synthesis: The Source of Insulin in Cell Clusters Differentiated from Murine Embryonic Stem Cells
Hyun Joon Paek,
Jeffrey R. Morgan,
Michael J. Lysaght
Center for Biomedical Engineering, Brown University, Providence, Rhode Island, USA
Key Words. Diabetes • Insulin • Embryonic stem cell • Glucose stimulation • Immunostaining
Correspondence: Hyun Joon Paek, Ph.D., Center for Biomedical Engineering, Brown University, Providence, Rhode Island, USA. Telephone: 401-863-3262; Fax: 401-863-1753; e-mail: Hyun_Paek{at}brown.edu
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ABSTRACT
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The source of insulin released from insulin-releasing cell clusters (IRCCs) differentiated from embryonic stem cells remains unclear. Rajagopal et al. have suggested that IRCCs do not synthesize but secrete insulin that had been absorbed from media during the multistep protocol. We report here further data relevant to this controversy. No radioisotopic labeling of insulin was observed when IRCCs were incubated in a medium containing 35S-cysteine. Less than 1% of the extra-cellular stoichiometric C-peptide equivalent to insulin was secreted during glucose stimulation. However, intracellular immunostaining and immunogold labeling were both positive for C-peptide. Finally, a mass balance calculation showed that simple equilibration of IRCCs by Fickian diffusion from media accounted for at most 4% of secreted insulin. These findings and further analysis of the results of others suggest that the mechanism of insulin secretion by IRCCs is a combination of sequestration and de novo synthesis.
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INTRODUCTION
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The utility of pancreatic islet-like cells derived from embryonic stem cells (ESCs) in cell-replacement therapy for type 1 and type 2 diabetes remains controversial [1–10]. Soria and colleagues [11, 12] originally derived insulin-producing cells from murine ESCs by using cell-trapping technology and demonstrated normalization of blood glucose in streptozotocin-induced diabetic mice. The following study by Lumelsky et al. [1], which manipulated culture conditions to derive insulin-releasing cell clusters (IRCCs) from murine ESCs without genetic modification, has been a subject of recent debate due to the presence of high-concentration exogenous insulin in the media used for the differentiation process and absence of pancreatic duodenal homeobox1 (Pdx-1) expression. In reproducing the protocol of Lumelsky et al. [1], Rajagopal et al. [2] did not detect expression of messenger RNA (mRNA) for insulin 1 and detected only weak expression of insulin 2 mRNA. The absence of insulin 1 could be indicative of ectodermal origin of IRCCs rather than endodermal, for ectoderm expresses only insulin 2. IRCCs cultured with fluorescein isothiocyanate (FITC)–labeled insulin during differentiation showed positive immunostaining for insulin with a rhodamine-conjugated antibody. However, both FITC and rhodamine were colocalized, suggesting that the positive insulin staining observed was due to the presence of FITC-labeled insulin. To explain such results, Rajagopal et al. [2] proposed that insulin secreted by IRCCs came from the media in which they had been cultured, not from de novo synthesis. In a separate series of experiments, Hori et al. [3] maintained normoglycemia in streptozotocin-induced nonobese diabetic severe-combined immunodeficient mice over 3 weeks using implanted IRCCs. Glucose responsiveness of IRCCs was demonstrated through glucose tolerance testing of murine recipients. Hyperglycemia returned immediately after explantation of IRCCs [3]. Insulin stored from sequestration of exogenous insulin is unlikely to lead to such observations, for secretion of exogenous insulin will diminish over time. More recently, Segev et al. [4] demonstrated further differentiation and increased synthesis and secretion of insulin by IRCCs by adding a step of suspension culture at the end to the protocol of Lumelsky et al. [1]. Furthermore, they detected expression of insulin mRNA, Pdx-1, and prohormone convertases by reverse transcription–polymerase chain reaction [4]. Segev et al.’s [4] results are thus consistent with de novo synthesis of insulin.
ESCs differentiation is a complex process that could be influenced in an unpredictable fashion by small changes in culture conditions [2–4]. Nevertheless, the findings of different groups are difficult to reconcile. Resolution of these inconsistencies and varying perspectives over the source of insulin in IRCCs is all the more important given the current policy debates over the utility and ethics of ESC research.
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MATERIALS AND METHODS
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Cells
An embryonic stem D3 line, derived from a strain of 129/Sv+C/+P mouse fibroblasts, was obtained from American Type Culture Collection (ATCC; Manassas, VA, http://www.atcc.org) and expanded for 2 months before study according to the ATCC protocol. During expansion, cells were cultured on a mitotically inhibited mouse embryonic fibroblast feeder layer in knockout Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Grand Island, NY, http://www.invitrogen.com) supplemented with 15% ESC-certified fetal bovine serum (FBS), 2-mercaptoethanol (2-ME) (Invitrogen), and leukemia inhibitory factor (LIF) (ESGRO, Chemicon, Temecula, CA, http://www.chemicon.com). Excess ESCs were stored in liquid nitrogen for later use.
IRCC Derivation
IRCCs were derived from murine ESCs according to the protocol of Lumelsky et al. [1] as modified by Hori et al. [3]. This protocol comprises a five-stage process using different culture conditions for each stage. During stage 1, ESCs were expanded on mouse embryonic fibroblast feeder layer in knockout DMEM supplemented with 15% ESC-certified FBS, LIF, 2-ME, nonessential amino acids, and L-glutamine (Invitrogen). In stage 2, ESCs were then trypsinized, transferred into ultra-low-attachment six-well plates (Corning, Acton, MA, http://www.corning.com), and cultured as a suspension to spontaneously form embryoid bodies (EBs) and initiate differentiation. During stage 3, EBs were collected and plated in 60-mm tissue culture dishes (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com), and the nestin-positive neural progenitor cells among EBs were selected using serum-free insulin-transferrin-selenium-fibronectin medium. Surviving selected cells were trypsinized and reseeded in six-well plates previously coated sequentially with poly-L-ornithine and fibronectin (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). They were proliferated in modified N2 medium supplemented with a mitogen, basic fibroblast growth factor (R&D Systems, Minneapolis, http://www.rndsystems.com), and B-27 supplement (Invitrogen) to enhance proliferation of the cells. During stage 5, the expanded cells were induced to differentiate into IRCCs in the modified N2 medium with a PI3K inhibitor, Ly294002 (Calbiochem, San Diego, http://www.emdbiosciences.com/html/CBC/home.html) and nicotinamide (Sigma). Media used in stages 4 and 5 also contained insulin (Invitrogen) at a concentration of 25 µg/ml. B-27 supplement also contained insulin, but its contribution to the total insulin content in stage 4 medium was negligible (40 pg/ml).
Radioisotopic Labeling
Control rat islets and IRCCs were cultured under conditions in which newly synthesized insulin would contain radiolabeled cysteine and thus could be distinguished from supplemented insulin in the medium. Following preincubation in cysteine-free DMEM (labeling medium) for 15 minutes to eliminate endogenous cysteine, cells were incubated at 37°C for 6 hours in labeling medium supplemented with 500 µCi [35S]cysteine (PerkinElmer, Boston, http://www.perkinelmer.com). Supernatant was collected. Cells were then separated and lysed in 250 µl buffer containing 25 mM sodium tetraborate (Na2B4O7), 3% bovine serum albumin (BSA), 1% Tween-20, 1 mM phenylmethanesulfonyl fluoride, 0.1 mM E-64, 1 mM EDTA, and 0.1% sodium azide (Sigma). Lysates were centrifuged at 13,000g for 5 minutes, and supernatant was collected.
Immunoprecipitation and Autoradiography
Rat islet supernatant (control), IRCC supernatant from 35S-cysteine–labeled cells, and IRCC lysates from 35S-cysteine–labeled cells were collected. One hundred microliters of each sample was incubated with 100 µl dilution buffer containing 0.1% Triton X-100 (Bio-Rad Laboratories, Hercules, CA, http://www.bio-rad.com) and 0.1% bovine hemoglobin (Sigma) in Tris/saline/azide (TSA) solution and 15 µl rabbit anti-rat insulin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com) or 15 µl of normal rabbit immunoglobulin G (IgG) on a shaker overnight at 4°C. On the following day, 20 µl of a protein A–agarose bead (Santa Cruz) suspension was added to the mixture and incubated overnight again on a shaker at 4°C. After incubation, the mixture was centrifuged and the supernatant was discarded. Beads were then washed twice with 1 ml dilution buffer, once with 1 ml TSA buffer containing 0.01 M Tris·Cl, 0.14 M sodium chloride, and 0.025% sodium azide in milli-Q water at pH 8.0, and once with 1 ml 0.05 M Tris buffer sequentially. After the last wash, beads were resuspended in 30 µl loading buffer containing 20% 0.5 M Tris·Cl buffer, 24% glycerol, 10% sodium dodecyl sulfate (10% SDS) (Sigma), 2% 2-ME, and 4% Coomasie G-250 (0.5% solution, Bio-Rad) and boiled for 5 minutes. Total secreted proteins by rat islets and IRCCs and proteins eluted from the agarose beads into loading buffer were electrophoresed on 16.5% Tristricine gel. Gels were dried, placed on BioMax Transcreen LE-enhancing screen, underneath which Biomax MS x-ray film (Eastman Kodak, Rochester, NY, http://www.kodak.com) was inserted, and incubated for 7 days at – 80°C for autoradiography.
Glucose-Stimulated Release of Insulin and C-Peptide
Upon completion of stage 5, 50 IRCCs were handpicked and transferred to Krebs-Ringer bicarbonate (KRB) buffer (Sigma). After an initial 30-minute basal stimulation at 10 mM glucose, IRCCs were incubated in 25 mM glucose solution in KRB buffer for 1 hour. Samples were taken at 30 minutes (basal) and 90 minutes (stimulated). Insulin concentration in each sample was determined using an enzyme-linked immunosorbent assay (ALPCO Diagnostics, Windham, NH, http://www.alpco.com). Radioimmunoassay (RIA) (Linco Research, St. Charles, MO, http://www.lincoresearch.com) based on guinea pig anti-rat C-peptide antibody, cross-reacting with the murine counterpart according to the manufacturer, was used to measure C-peptide concentration. Controls were run with handpicked rat islets. Stoichiometric equivalents were calculated based on average molecular weights of 5,796 Da and 3,834 Da for insulin and C-peptide, respectively.
Immunocytochemistry
Stage 5 IRCCs were plated in chamber slides coated with poly-L-ornithine and fibronectin. Cells were fixed in methanol for 2 minutes and treated with 5% BSA (Sigma) and 10% goat serum (Jackson Immuno Research Laboratories, West Grove, PA, http://www.jacksonimmuno.com) in phosphate-buffered saline (PBS). Guinea pig anti-rat C-peptide antiserum (Linco Research) diluted at 1:250 was the primary antibody. Rhodamine-conjugated goat anti–guinea pig IgG (Chemicon) diluted at 1:500 was the secondary antibody and was used to visualize primary antibody binding. Controls for nonspecific binding included (a) normal guinea pig IgG at 1:250 along with the secondary antibody at 1:500 and (b) secondary antibody at 1:500 dilution. A Zeiss Axiovert 200M fluorescent microscope (Carl Zeiss, Hallbergmoos, Germany, http://www.zeiss.com) was used to observe each staining.
Immunogold Labeling and Transmission Electron Microscopy Analysis
IRCCs were fixed in 1% glutaldehyde, 0.2% picric acid (Electron Microscopy Science, Ft. Washington, PA, http://www.emsdiasum.com) for 30 minutes at 4°C, dehydrated, and embedded in resin. IRCCs were then sectioned on a Reichert-Jung Ultracut E, resulting in 80- to 85-nm-thick sections. Ultra-thin sections were sequentially treated with blocking buffer, as described in immunocytochemistry, and incubated overnight at 4°C with guinea pig anti-rat C-peptide antiserum or normal guinea pig IgG; control diluted at 1:500. After washing in PBS, the sections were then treated with gold-labeled secondary antibody (10-nm gold particles, goat anti–guinea pig IgG; Electron Microscopy Science) diluted at 1:20. The cells were then stained with lead and uranyl acetate. Stained sections were viewed on a 410 transmission electron microscope (Philips, Holland, http://www.philips.com).
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RESULTS
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IRCCs were successfully derived using the protocol [3] described above. The morphology of cells in each stage was similar to that reported by others.
Figure 1
shows the autoradiography print of the SDS-PAGE of insulin produced by islets and IRCCs cultured in medium containing 35S-cysteine. Tracks are shown for islet supernatant (lanes 1–3), IRCC supernatant (lanes 4–6), and IRCC lysates (lanes 7–9). Lanes 1, 4, and 7 represent total protein; lanes 2, 5, and 8 are from a precipitate of protein with rabbit anti-rat insulin antibody; and lanes 3, 6, and 9 show a control precipitate with a nonspecific primary antibody control, normal rabbit IgG. Autoradiography clearly showed bands for insulin and B-chain of insulin for rat islet-positive control (Fig. 1; lanes 1 and 2). Lane 3 shows bands in a high-molecular-weight range, which are a result of nonspecific binding with normal guinea pig IgG, although the identity of proteins is uncertain based only on their molecular weights. However, a combination of lanes 2 and 3 clearly demonstrates that insulin precipitation in lane 2 was not from nonspecific binding of normal rabbit IgG to insulin, because insulin bands from antibody-specific binding are not present in lane 3. In contrast, no detectable radiolabeled insulin was present in insulin secreted from IRCCs in either supernatant or the lysates (Fig. 1; lanes 4, 5, 7, and 8). Nonspecific binding of insulin to normal rabbit IgG is not observed in lanes 6 and 9. This is consistent with the position that IRCCs do not synthesize insulin de novo.
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Figure 1. Autoradiography of immunoprecipitated insulin synthesized from 35S-radiolabeled cysteine. Islet supernatant (lanes 1–3), IRCCs supernatant (lanes 4–6), IRCCs lysates (lanes 7–9). Total protein secreted (lanes 1, 4, and 7) serves as a positive control. Immunoprecipitation using rabbit anti-rat insulin anti-body (lanes 2, 5, and 8) is the variable under study. Immunoprecipitation using normal rabbit IgG (lanes 3, 6, and 9) is a negative control. The absence of bands for insulin and B-chain of insulin in lanes 5 and 8, in contrast to lane 2, confirms the lack of de novo insulin synthesis. The bands and dark shades in lanes 1, 4 and 7 indicate the successful uptake of 35S-cysteine into the cells. Abbreviations: IgG, immunoglobulin G; IRCCs, insulin-releasing cell clusters.
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Table 1 reports the quantities of insulin and C-peptide released along with predicted stoichiometric equivalence during glucose stimulation. C-peptide, released when proinsulin is cleaved to form insulin [13], is a convenient antigenic marker of insulin synthesis. In our hands, 10 IRCCs secreted approximately 9 ng of insulin during 1-hour stimulation in KRB buffer with 25 mM glucose, which was a rapid ninefold increase from the quantity of insulin secreted during 30-minute basal stimulation at 10 mM glucose. This is about 56% of the 16 ng released by 10 control islets and is comparable with quantities reported by Hori et al. [3]. The relative increase in quantity of insulin secreted by IRCCs in 25 mM glucose buffer might have been even greater if the cells were incubated in a lower concentration of glucose than 10 mM for basal stimulation [references]. Concentrations of C-peptide secreted along with insulin were measured by RIA with guinea pig anti-rat C-peptide antiserum. The quantity of C-peptide released along with the islets was only ~15% of the predicted equivalent, possibly due to the sensitivity of the RIA kit and the binding affinity of antibody used in this assay. The quantity of extracellular C-peptide secreted by IRCCs was <0.5% of the stoichiometric equivalent. These results are also consistent with release of stored insulin rather than de novo synthesis.
As shown in Figure 2, immunostaining and immunogold labeling were positive for intracellular C-peptide in ~50% of IRCCs. For immunogold labeling, clusters of more than two gold dots suggested the presence of C-peptide within the cytoplasm of IRCCs. Positive immunostaining and immunogold labeling were observed in 50% of IRCCs (Figs. 2A, 2B). Controls did not show any nonspecific immunostaining and exhibited single dots from immunogold labeling in less than 5% of the cells (Figs. 2C–2F). The presence of intracellular C-peptide is consistent with de novo synthesis. Interestingly, transmission electron microscopy, used for immunogold labeling, also revealed that IRCCs lacked well-developed secretory granules compared with rat islet controls.
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Figure 2. Immunostaining for intracellular C-peptide by (A) immunocytochemistry with guinea pig-derived anti-rat C-peptide primary antibody and (B) immunogold labeling using the same primary antibody (particles = 10 nm). Secondary antibody controls with either (C) rhodamine-conjugated goat anti-guinea pig IgG or (D) gold-labeled goat anti-guinea pig IgG. (E, F): Non-specific primary antibody control with normal guinea pig IgG and secondary antibodies. The orange color in (A) and dark gold dots (arrows) in (B) indicate the presence of at least some C-peptide in the IRCCs. Ultrastructure in (B) also reveals that IRCCs lack well-developed secretory granules. Abbreviations: C, cytosol; IgG, immunoglobulin G; IRCCs, insulin-releasing cell clusters; N, nucleus.
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Table 2 contains the details of a mass-balance calculation for sequestered insulin. Assuming Fickian diffusion, the quantity of insulin in IRCCs fully equilibrated with media can readily be calculated as the product of the volume of a single IRCC and the maximum concentration of insulin in the media to which the cells are exposed. The volume of a single IRCC was 1.4 x 106 µm3 (1.4 x 10–6 ml) based on the assumption that an IRCC was hemiellipsoidal in shape with major and minor axes (r1 = 150 µm, r2 = 75 µm, r3 = 60 µm) obtained from optical microscopy. The total volume of 50 IRCCs that were used in glucose stimulation was 7 x 107 µm3 (7 x 10–5 ml). At 100% equilibration with an insulin concentration of 25 µg/ml, 50 IRCCs would contain 1.7 ng of insulin. However, the quantity of insulin released during glucose stimulation by 50 IRCCs was 48 ng (Table 1
), 28 times greater than the amount they would contain if fully equilibrated with the highest concentration of insulin to which they had been exposed. If insulin was bound to intracellular macromolecules, and insulin is notoriously sticky, then concentration in the cell could exceed that in free solution. However, bound insulin is unlikely to be released upon glucose stimulation. This line of reasoning and our observation of the absence of well-developed secretory granules suggest that some mechanism other than sorption/release must be at play.
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DISCUSSION
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mRNA was not examined specifically in our study because previously published studies reported on expression of mRNA for insulin 1 and 2 using a similar protocol [3, 4]. We decided to report on methods that were not previously used, such as RIA, immunogold labeling for C-peptide, and uptake of 35S-labeled cysteine.
Taken together, some aspects of our data support de novo synthesis of insulin, whereas other portions are consistent with the hypothesis of sequestration and rerelease. Immunostaining qualitatively shows positive expression of C-peptide, the byproduct of insulin synthesis, within the cytosol of IRCCs, suggesting that at least some level of insulin synthesis is present within IRCCs. A mass balance calculation also demonstrates that the insulin sequestered by Fickian diffusion cannot account for all of the insulin secreted by IRCCs, even in the limiting case of apoptotic cells fully permeable to insulin in media. Thus, some degree of de novo insulin synthesis is required to explain our findings.
However, 35S-cysteine radioisotopic labeling and immunoprecipitation are unable to detect any significant quantity of newly synthesized insulin. The polyclonal antibody used for this study recognizes proinsulin as well as insulin and B-chain of insulin. However, a band corresponding to the molecular weight of pro-insulin was not observed on autoradiograph. However, this could also be indicative of a very low level of proinsulin synthesis under the detection level of immunoprecipitation and autoradiography.
RIA showed that virtually no extracellular C-peptide accompanies insulin released by IRCCs. If newly synthesized insulin is in a form of proinsulin, the antibody for C-peptide in the RIA kit used for this assay will not be able to recognize it. This assay may thus understate the intracellular C-peptide expression level that has been observed in immunostaining and immunogold labeling.
Although more complex hypotheses are possible, the simplest explanation for these findings is that both sequestration and synthesis are operative simultaneously. In this case, IRCCs indeed synthesize insulin, as suggested by Lumelsky et al. [1], Hori et al. [3], and Segev et al. [4], but also sequester and rerelease it, as suggested by Rajagopal et al. [2]. The quantity of insulin synthesized de novo is suspected to match the quantity of C-peptide detected by RIA.
A somewhat expanded and more speculative hypothesis arises when our findings are combined with previously published results of others [1–4]. Two-week exposure to a high concentration of exogenous insulin within the media during the last two phases of the derivation process may temporarily suppress de novo synthesis and secretion of insulin by IRCCs. Thus, the absence of messenger RNA observed by Rajagopal et al. [2] may be due to temporary suppression of insulin synthesis and not to the complete absence of capacity for insulin synthesis. After exogenous insulin stores are depleted, synthetic capacity may return, enabling the 3-week maintenance of normoglycemia in diabetic mice [3]. Moreover, the in vivo environment may provide IRCCs with unidentified growth factors and conditions, such as a three-dimensional environment, that further differentiate IRCCs, resulting in increased insulin synthesis. Although our suppression hypothesis is speculative, we are at a loss for a credible alternative explanation for all the extant data.
In principle, the question could be directly resolved by analyzing an amino acid sequence of secreted insulin, because bovine insulin in the media differs by four amino acids from the murine counterpart in IRCCs. However, quite large quantities of the secreted insulin would be required. Because rodent models to test the efficacy of transplanted islets are well established, a better alternative might be for groups to test the IRCCs derived by their specific protocol in vivo over time frames in which positive results would rule out sequestration and rerelease. Investigators could then examine a change in the level of insulin mRNA expression in IRCCs recovered from these protocols.
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CONCLUSION
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In this study, radioisotopic labeling of insulin by IRCCs incubated in a medium containing 35S-cysteine was insignificant, as was extracellular secretion of C-peptide. However, IRCCs stained positive for intracellular C-peptide, and a mass balance suggested that sequestered insulin could not account for the total quantity of insulin secreted during glucose stimulation. We conclude that the sources of insulin secreted by IRCCs include both sequestration from media and de novo synthesis by IRCCs, and we speculate that the former phenomenon may actively suppress the latter.
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ACKNOWLEDGMENTS
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We thank Dr. Edward Hawrot and Hilary Hartlaub for the radioactive facility used for radioisotopic labeling, immunoprecipitation, and autoradiography. We also thank Paula Weston for her assistance in immunogold labeling. This work was supported by Pierre Galletti Graduate Fellowship (Sorin Group, Via Crescentino, Italy, http://www.sorin-cid.com) and a seed grant from Brown University (Providence, RI, http://www.brown.edu).
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Received October 20, 2004;
accepted for publication April 25, 2005.
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Abstract
Introduction
