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EMBRYONIC STEM CELLS |
Transplantation Research Immunology Group, Nuffield Department of Surgery, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom
Key Words. ESC • Differentiation • Insulin-secreting cells • In vitro differentiation • Cell transplantation • Pancreatic differentiation • ES cells • Diabetes
Correspondence: Correspondence: Ashleigh S. Boyd, D.Phil., Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, United Kingdom. Telephone: +44 1865 275 606; Fax: +44 1865 275 515; e-mail: ashleigh.boyd{at}path.ox.ac.uk
Received on September 11, 2007;
accepted for publication on February 18, 2008.
First published online in STEM CELLS EXPRESS March 6, 2008.
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
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Disclosure of potential conflicts of interest is found at the end of this article.
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
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To clarify the potential of mouse ESCs to generate IPCCs, we modified, compared, and contrasted three protocols from Lumelsky et al. [2], Hori et al. [4], and Blyszczuk et al. [14]. Using immunofluorescence and qualitative and quantitative reverse-transcriptase-PCR, we systematically characterized the morphological and pancreatic endocrine-like features of IPCCs. Of particular note, we determined de novo insulin synthesis from IPCCs by assessment of c-peptide expression, a by-product of insulin synthesis [15]. In addition, glucose stimulation/insulin secretion assays were deployed to define the ability of ES-cell derived IPCCs to release insulin in response to glucose stimulation. Finally, the in vivo functionality of IPCCs was ascertained using transplantation of IPCCs into mice that were rendered diabetic by the β cell toxin streptozotocin.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
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ESC Lines and Maintenance of ESCs
The embryonic stem cell lines used for this project, designated ESF 122 (H2k), ESF 134 (H2b), and ESF 150 (H2d), were a kind gift from Dr. Frances Brook and Professor Richard Gardner at the Department of Zoology, University of Oxford (see supplemental online Table 1). A sample containing 1 x 106 ESCs was plated into 25-cm2 flasks containing a confluent layer of mitotically inactivated primary embryonic fibroblasts (3,000 rad) in ESC medium composed of knockout Dulbecco's modified eagle medium (KO-DMEM) (Invitrogen, Paisley, Scotland, http://www.invitrogen.com), 15% FCS, 1% 100 µM L-glutamine, 1% nonessential amino acids (non-eAAs) (all Invitrogen), 1% 100 µM penicillin-streptomycin, 100 µM β-ME, and 100 µl/10 ml medium 10 µg/ml LIF (Chemicon, Temecula, CA, http://www.chemicon.com).
Differentiation of ESCs into IPCCs
Three previously published methods were utilized in the attempt to differentiate ESCs into IPCCs. These methods shall be referred to throughout as the Hori [4], Lumelsky [2], and Blyszczuk [14] methods, respectively. The medium used for each stage is detailed in supplemental online data.
Stage I, generation of embryoid bodies (EBs), was achieved by the hanging drop method, which entailed preparing ESC suspensions at a density of 24,000 cells/ml or 600 cells/20 µl drop in ESC medium. Approximately 60 drops were placed onto the base of a 100-mm2 tissue-culture dish. The lid was placed on and the dish inverted in one quick motion to ensure droplets remained in place. Two days later, the plates were turned right side up and flooded with ESC medium to suspend the drops (stage II). Two hanging drop culture plates were combined and plated into one 10-cm2 culture plate. At this stage the protocols diverged.
Generation of cell clusters using the Lumelsky/Hori method: Stage II EBs remained on the 100-mm2 dish for 7 days. EBs were approximately 150–200 µm. After 7 days, the EBs were trypsinized and transferred to a 60-mm2 tissue-culture plate for stage III of the differentiation. The medium was changed 2 days later after the cell bodies had attached to the culture dish, at which point serum-negative insulin transferrin selenium fibronectin medium was added. After 7 days, the cells were trypsinized and transferred to a plate coated with poly-L-ornithine (PLO) + fibronectin for stage IV. For 1 day of culture at this stage, the culture medium contained 15% serum. After 1 day, the cells received N2 medium containing bFGF and B27 supplement. For stage V, 1 week later the N2 + bFGF + B27 medium was changed to N2 medium containing nicotinamide and either B27 (stage VNB medium, Lumelsky protocol) or Ly294002 (stage VNL medium, Hori protocol). Cells were harvested after growing for a minimum of 1 week in this medium.
Generation of cell clusters using the Blyszczuk method (stages III and IV): At day 5 + 1, the contents of one culture plate (approximately 50–60 EBs) were transferred to a gelatin-coated 60-mm plate. At day 5 + 9, EBs were dissociated using 0.1% trypsin:0.08% EDTA solution. Approximately 0.5 ml cell suspension was added to 60-mm plates precoated with PLO and laminin in B2 medium. Cell clusters were harvested after a minimum of 19 days culture at this stage.
Immunofluorescence
Samples were harvested, embedded in OCT Tissue-Tek (Miles Diagnostics, Elkhart, IN, http://www.mileslabs.com) compound, and frozen in liquid nitrogen until processed. Sections (6 µm), made on a cryostat, were air-dried overnight and then fixed in acetone for 15 minutes at 4°C. Sections were blocked with phosphate-buffered saline (PBS)/4% goat serum and incubated with the primary antibody diluted in the same blocking solution overnight at 4°C. Primary antibodies used were guinea pig anti-swine insulin (1:300; DakoCytomation, Glostrup, Denmark, http://www.dakocytomation.com) and rabbit anti-human C-peptide (1:150; MorphoSys AG [formerly Biogenesis Ltd], Martinsried/Planegg, Germany, http://www.morphosys.com). The next day, sections were washed and incubated first with the C-peptide secondary antibody, Alexafluor-564 conjugated goat anti-rabbit (1:150; Molecular Probes, Eugene, OR, http://probes.invitrogen.com), for 1 hour at room temperature before washing and application of the insulin secondary antibody for 1 hour at room temperature (FITC rabbit anti-guinea pig, 1:150; DakoCytomation). After further washing, the sections were mounted in Vectashield medium containing 4',6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Peterborough, UK, http://www.vectorlabs.com) and examined with a Zeiss fluorescence microscope using Openlab 4.0.1 software (Agilent Technologies, Palo Alto, CA, http://www.agilent.com).
Glucose Stimulation Assays
Insulin released by IPCCs in response to glucose stimulation was measured with an anti-mouse insulin ELISA kit (Mercodia, Uppsala, Sweden, http://www.mercodia.com) according to the manufacturer's instructions. For the assay, each 60-mm2 dish of cells was washed with PBS and the plate incubated at 37°C for 1 hour first with 2 ml 3.3 mmol/l glucose solution (Sigma-Aldrich Company Ltd, Poole, UK, http://www.sigmaldrich.com) and then subsequently with 25 mmol/l glucose solution (Sigma-Aldrich). The supernatants were harvested after each stimulation and subjected to ELISA. To determine total protein content, the cells on each plate were disrupted using a protein lysis buffer prepared with 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 5 mM EDTA, 1.5% Triton-X 100, 4 mM phenyl methylsulfonylfluoride, and 10 mM dithiothreitol. Total protein from lysed cells was measured by a standard protein assay using a BCA Protein Assay kit (Perbio Science Ltd, Cramlington, UK, http://www.perbio.com) using a spectrophotometer at wavelength 562 nm.
RT-PCR and Q-PCR
Total cellular RNA was isolated from undifferentiated ESCs, end-stage IPCCs, pancreas, islets, and spleen using the Stratagene Absolutely RNA kit. RNA (2 ng) was reverse transcribed into cDNA with murine Maloney leukemia virus (MMLV)-reverse transcriptase (RT, Invitrogen) according to the manufacturer's instructions. cDNA (1–2 µl) was amplified for 35 cycles by PCR on a gradient cycler using gene-specific oligonucleotide primers for the following genes: amylase-2, glucagon, insulin-1, insulin-2, neurogenin-3 (Ngn-3), and pancreatic-duodenal homeobox-1 (Pdx-1). Primer sequences and PCR conditions for each gene are provided in supplemental online Table 2. Per sample, the PCR reaction mixture contained 0.3 µl Taq polymerase (Bioline, London, http://www.bioline.com), 1 µl 20 µM dNTPs, 1.5 µl MgCl2 (Bioline), 2.5 µl 10x reaction buffer (Bioline), 16.7 µl distilled water, 1 µl each of the sense and antisense primers (10 µM stock, MWG Biotech, Ebersberg, Germany, http://www.mwg-biotech.com), and 1–2 µl cDNA. PCR reactions were subjected to electrophoresis on 1.5–2% agarose (Seachem Laboratories, Inc., Madison, GA, http://www.seachem.com) gels containing 10 µg/ml ethidium bromide to visualize the PCR products.
For quantitative real-time PCR (Q-PCR), 2.5 ng of template was used, and for each sample, PCR was performed in duplicate. The following TaqMan Assays-On-Demand primer and probe sets were used for Q-PCR analysis: amylase-2, glucagon, insulin-1, insulin-2, and Pdx-1 (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com). Test samples were analyzed in comparison to HPRT on an ABI analyzer (Applied Biosystems) and quantitation achieved using the standard two x delta CT method. RT samples and negative controls (no template) were run together with test samples, and standard curves were used for each gene tested to analyze the efficiency of the PCR reaction.
Induction of Experimental Diabetes
Diabetes was chemically induced 7 days prior to transplantation with a single intraperitoneal injection with the beta cell toxin streptozotocin (250 mg/kg) (Sigma-Aldrich).
Transplantation of IPCCs
For transplantation of IPCCs, mice were anesthetized with 300 µl per animal by subcutaneous (s.c.) injection of buprenorphine (Vetergesic, Alstoe Ltd, Melton Mowbray, UK, http://www.alstoe.com) and 300 µl/animal of a 1:1 mix of medetomidine hydrochloride (Domitor, Pfizer, New York, http://www.pfizer.com) and ketamine hydrochloride (Ketaset, Pfizer). The lower left of each animal's abdomen was swabbed with ethanol and shaved to expose the skin. A vertical incision was made through the dermal layers and peritoneum, and the left kidney was exposed. An incision was made on the kidney surface to open the subcapsular renal space, and the IPCCs were inserted under the capsule. Post-transplantation 4/0 vicryl sutures were used to close the peritoneum and the skin before administration of 300 µl of the reversal agent atipamezole hydrochloride (Antisedan, Pfizer) sc to each animal.
Isolation and Transplantation of Pancreatic Islets
Islets were isolated by collagenase digestion of the pancreas, followed by centrifugation through a discontinuous Ficoll gradient, as previously described [16, 17]. Islets were transplanted using the same procedure as for IPCCs.
Blood Glucose Measurements
Blood glucose was measured with an AccuChek Advantage Glucosemeter (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com) using blood from a single needle prick to the tail.
Statistical Analysis
Statistical analysis was performed using the Student's t test.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
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To enable a relative quantitative evaluation of the endocrine potential of the different protocols, Q-PCR for insulin-1, insulin-2, Pdx-1, glucagon, and amylase-2 mRNA was performed on each of the protocols, undifferentiated ESCs, and pancreatic islets (n = 3 experiments). The relative mRNA expression of each of the pancreatic genes was appreciably higher in pancreatic islets compared with IPCCs derived from any of the three protocols (data not shown). Although relative parity was observed in amylase-2 and Pdx-1 mRNA expression across all protocols, the Blyszczuk protocol displayed superior relative expression of insulin-1 and insulin-2 mRNA compared to the Hori and Lumelsky protocols; of particular note, Blyszczuk IPCCs expressed approximately 18-fold more insulin-1 mRNA than IPCCs generated from either the Hori or the Lumelsky protocols (Fig. 2D). Additionally, Blyszczuk IPCCs expressed approximately 13-fold higher insulin-2 mRNA than Hori IPCCs and approximately 5-fold higher insulin-2 mRNA than Lumelsky IPCCs (Fig. 2D). Furthermore, insulin-2 mRNA expression from Lumelsky IPCCs was approximately 2-fold greater than that from Hori IPCCs, and glucagon mRNA expression was marginally elevated in Hori IPCCs compared with Lumelsky IPCCs (Fig. 2D). These data jointly demonstrate that the Blyszczuk protocol forms IPCCs with a greater pancreatic endocrine molecular signature than either the Hori or the Lumelsky protocols.
Comparison of Insulin, c-Peptide, and Glucagon Expression in IPCCs by Immunofluorescence Microscopy
Using immunofluorescence microscopy, further characterization of ESF 122-derived IPCCs was performed at the protein level for insulin and c-peptide, a molecule released upon cleavage of the insulin precursor pro-insulin [18]; c-peptide thus allows cells that have adsorbed insulin from the culture medium [10] to be discerned from those that synthesized insulin de novo. Insulin and glucagon costaining was also performed. Glucagon is produced by 
cells located around the periphery of an islet, enclosing the central core of β cells. Importantly, colocalization of each of these antigens with the nuclear dye DAPI was used to distinguish authentic patterns of immunofluorescence staining. Sectioned pancreas from adult mice of the same strain as ESF 122 served as positive controls, and these sections demonstrated definitive coexpression of the aforementioned hormones (Fig. 3).
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Blyszczuk clusters stained strongly for insulin, glucagons, and DAPI (Fig. 3B). In contrast, Hori and Lumelsky clusters presented diminished signals for insulin and glucagons, respectively, and exhibited a similar pattern of staining in terms of cellular coexpression of insulin, glucagons, and DAPI (Fig. 3B). From these data, we infer that IPCCs generated using the Blyszczuk protocol displayed a robust pancreatic phenotype compared to IPCCs generated using either the Hori or the Lumelsky protocols.
Glucose Stimulation of Putative IPCCs In Vitro
To determine whether IPCCs could release insulin in a glucose-dependent manner and were able to function in a manner akin to pancreatic β cells, IPCCs generated by the Hori, Lumelsky, and Blyszczuk protocols were next assessed for their ability to secrete insulin in response to sequential stimulation with a low (3.3 mmol/l) and a high (25 mmol/l) concentration of glucose. After stimulation, the supernatants were analyzed by ELISA to detect the presence of insulin. IPCCs differentiated by the Lumelsky or the Blyszczuk method released more insulin than Hori-generated cells in response to 3.3 mmol/l glucose concentration. The highest level of insulin secretion was approximately 15 µg/l by cell clusters derived from the ESF 134 cell line, differentiated by the Lumelsky method. Out of 16 Blyszczuk or Lumelsky samples derived from 3 ESC lines, 8 samples were able to secrete more than 6 µg/l insulin in response to glucose (Fig. 4A and data not shown). Blyszczuk-generated IPCCs performed in a similar manner to the Lumelsky IPCCs (Fig. 4A). The highest levels of insulin secretion in all protocols were observed in response to the 3.3 mmol/l glucose concentration supernatants. These data show that Hori-, Lumelsky-, and Blyszczuk-derived IPCCs contain insulin and release it upon stimulation with a low dose of glucose.
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Functionality of IPCCs In Vivo
Having assessed gene and protein expression and insulin release in each protocol, we next investigated the functional capacity of Blyszczuk, Hori, and Lumelsky IPCCs to rescue diabetes in vivo. A single intraperitoneal injection of the N-nitroso derivative of glucosamine, streptozotocin (STZ), was used to induce diabetes by directly destroying pancreatic β cells [19]. IPCCs or islets were transplanted into the subcapsular renal space of syngeneic diabetic mice 1 week following STZ challenge, and recipient blood glucose was monitored to assess glycemia (Fig. 5A).
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Removal of an engrafted kidney in recipients that were normoglycemic for more than 20 days caused a dramatic and instantaneous reversion to hyperglycemia, directly demonstrating that Blyszczuk IPCCs were responsible for the reversal of diabetes (n = 2 recipients; blood glucose levels of 32.4 and 34.9 mM were observed in these recipients after this procedure was performed). Importantly, sham transplantation, transplantation of undifferentiated ESCs, and day 10 EBs failed to rescue STZ-induced hyperglycemia at 72 hours after transplantation (A.S.B., unpublished observations). Jointly, these data show that only Blyszczuk IPCCs have the limited potential to rescue experimental diabetes in vivo. In addition, recipients developed teratomas after 2 to 3 weeks following transplantation with Blyszczuk IPCCs, as evidenced by grafts that were removed for histological examination (Fig. 5F).
To investigate why Blyszczuk IPCCs possessed less in vivo functionality than the equivalent number of syngeneic pancreatic islets, we examined whether the viability of IPCC grafts was compromised with time after transplantation. In all, 300 syngeneic Blyszczuk IPCCs were transplanted under the kidney capsule using nondiabetic syngeneic mice and their viability was examined post-transplantation. Recipients were sacrificed at day 3 and at day 9 post transplantation for a postmortem analysis of the graft site (n = 2 recipients for each time point). Hematoxylin and eosin staining of samples from day 3 recipients (Fig. 6A, 6B) revealed healthy-looking cell clusters. However, at day 9 the condition of the transplanted IPCCs had deteriorated. In many parts of the graft site, the clusters appeared necrotic, displaying perturbed cell membranes and insubstantial nuclei (Fig. 6C, 6D), especially those cells at the outer edge of the graft, under the capsule. These data show that IPCC graft viability was maintained over a short time frame and suggest that the inconsistent capacity of Blyszczuk IPCCs to maintain long-term normoglycemia may, in part, be ascribed to a decline in the viability of IPCCs in vivo.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
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IPCCs derived from the Blyszczuk, Hori, and Lumelsky protocols were first subjected to qualitative reverse transcriptase-PCR analysis to define whether they expressed pancreatic hormones and transcription factors including insulin-1, insulin-2, amylase-2, glucagon, and Pdx-1. Whether the gene expression seen is attributable to a single cell within the isolated cluster or whether all cells in that cluster have transcribed the gene has yet to be defined. This caveat aside, certain patterns of gene expression were observed in IPCCs generated by each of the three protocols, indicating a mixed pancreatic phenotype. Using quantitative PCR, we demonstrated that expression of insulin-1 and insulin-2 mRNA was consistently higher in Blyszczuk IPCCs compared to either Hori- or Lumelsky-generated clusters, implying that the Blyszczuk protocol was capable of generating IPCCs with superior de novo insulin-producing activity. Amylase-2 expression was also noted in all IPCCs, suggesting that the protocols may promote exocrine differentiation. This contrasts with the original reports by Lumelsky et al. [2] and Hori et al. [4]. However, another group attempting to recapitulate Lumelsky's protocol to generate IPCCs observed amylase expression [20], as have other groups generating IPCCs by other methods [6].
Using immunofluorescence to document coexpression of insulin and c-peptide in IPCCs, we observed an attenuated relative ratio of c-peptide to insulin in each of the protocols, suggesting that the insulin content of IPCCs is an unequal combination of de novo synthesis and adsorption from the culture medium [10]. Contrary to previous reports, the Lumelsky protocol did exhibit some c-peptide immunoreactivity, indicating a nominal level of de novo insulin synthesis. Most notably, however, the Blyszczuk protocol consistently produced the highest level of c-peptide out of the three protocols tested, indicating that this protocol was capable of superior de novo synthesis of insulin.
Glucagon was selected as a negative marker of β cell development on the premise that it is a hormone produced exclusively by cells within the islets [21]. In line with the gene-expression studies, prominent glucagon costaining with insulin in IPCCs was common to all three differentiation protocols, with the highest expression observed in the Blyszczuk protocol. Thus, IPCCs derived from ESCs may not be surrogate β cells but may, in contrast, more closely resemble a hybrid of and β cells. Glucagon/insulin costaining may alternatively indicate that IPCCs derived from ESCs are developmentally immature endocrine cells. Further studies should be directed at examining how glucagon coexpression influences the functionality of IPCCs.
Transplantation of Blyszczuk IPCCs was performed to functionally test their ability to reverse experimentally induced diabetes. In comparison to islet transplantation, however, comparable numbers of IPCCs were less able to provide long-term rescue from diabetes. Several potential contributory mechanisms may explain the inability of IPCCs to function long term in vivo. Shortly after transplantation (>3 days), we observed large areas of dead or dying cells on excised IPCC grafts. Because a large number of cell clusters had been transplanted into the small subcapsular space, some of the clusters may have been denied direct contact with the kidney or indeed the renal blood supply. On a related note, the size of the IPCCs may also be an issue in transplantation; the average cell cluster diameter in this system was 1.5–2 mm, almost eightfold higher than a mouse islet (A.S.B., unpublished observations). Thus, overall, the transplanted IPCCs may have been starved of nutrients and failed to survive in vivo.
Another reason for the inconsistent ability of IPCCs to reverse experimental diabetes may be ascribed to the developmental potential of these clusters. Two lines of evidence suggest that IPCCs have yet to complete their functional maturation into pseudoislets. As alluded to previously, glucagon and insulin costaining in the IPCC protocols may be indicative of an immature or developmental early endocrine cell. Secondly, expression of neurogenin-3 (Ngn3), which is observed throughout islet development but is downregulated in fully mature islets [22], was noted at the published end point of the protocols (A.S.B., unpublished observations).
IPCCs may also be defective in their glucose-sensing capacity in vivo. This contention is supported by the glucose stimulation assays that showed that IPCCs generated by each of the protocols released insulin in response to minimal glucose stimulation (3.3 mmol/l glucose) yet did not release significant amounts of insulin at higher glucose stimulation (25 mmol/l glucose). IPCCs may therefore merely release all the insulin from their cytoplasm after stimulation with the low dose of glucose and have no insulin reserve to release upon restimulation with a higher dose of glucose. Alternatively, the time taken by IPCCs to synthesize proinsulin and process insulin may exceed the 1-hour time span in which the cells were incubated with the higher dose of glucose. Thus, IPCCs may have released all of their intracellular insulin and had insufficient time to resynthesize more insulin for release during the high glucose incubation. Furthermore, IPCCs may have been capable of insulin production, but the hyperglycemia induced by a 25 mmol/l glucose period could have been toxic to the cells, causing them to cease to function. Finally, the number of IPCCs used may also be a determinant of their glucose-sensing capacity. This idea is supported by the glucose stimulation assays of islets that displayed defective sensing capacity at low cell number and glucose dose. However, at higher islet numbers, normal glucose-sensing ability was restored.
Transplantation of undifferentiated ESCs causes the formation of teratomas [23]. Moreover, when differentiated ESCs are transplanted, teratomas have also been observed, an observation that was recapitulated in this study. A plausible explanation for such a finding may be that ostensibly differentiated ES-derived IPCCs contain a proportion of undifferentiated ESCs. These stem cells could be removed from IPCCs by using a cell-sorting-based approach to remove cells expressing pluripotent stem cell markers, such as stage-specific embryonic antigen-1 (SSEA-1) [24], which are expressed only on mouse embryonic stem cells and not on their mature progeny. To further diminish the tumorogenic potential of IPCCs separated by this method, a negative cell selection may also be used for potential tumor cell antigens. For example, epithelial cell adhesion molecule (EpCAM) may be a germane marker that could be used for this purpose [25]. By using these collective cell-selection strategies to remove cells with tumorogenic potential, the long-term function of transplanted ESC-derived clusters may be directly or indirectly enhanced.
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CONCLUSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
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ACKNOWLEDGMENTS |
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REFERENCES |
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