Objectives: In diabetes mellitus type 1, beta cells are mostly destroyed; while in diabetes mellitus type 2, beta cells are reduced by 40% to 60%. We hope that soon, stem cells can be used in diabetes therapy via pancreatic beta cell replacement. Induced pluripotent stem cells are a kind of stem cell taken from an adult somatic cell by “stimulating” certain genes. These induced pluripotent stem cells may be a promising source of cell therapy. This study sought to produce isletlike clusters of insulin-producing cells taken from induced pluripotent stem cells.
Materials and Methods: A human-induced pluri-potent stem cell line was induced into isletlike clusters via a 4-step protocol, by adding insulin, transferrin, and selenium (ITS), N2, B27, fibroblast growth factor, and nicotinamide. During differentiation, expression of pancreatic β-cell genes was evaluated by reverse transcriptase-polymerase chain reaction; the morphologic changes of induced pluripotent stem cells toward isletlike clusters were observed by a light microscope. Dithizone staining was used to stain these isletlike clusters. Insulin produced by these clusters was evaluated by radio immunosorbent assay, and the secretion capacity was analyzed with a glucose challenge test.
Results: Differentiation was evaluated by analyzing the morphology, dithizone staining, real-time quantitative polymerase chain reaction, and immunocytochemistry. Gene expression of insulin, glucagon, PDX1, NGN3, PAX4, PAX6, NKX6.1, KIR6.2, and GLUT2 were documented by analyzing real-time quantitative polymerase chain reaction. Dithizone-stained cellular clusters were observed after 23 days. The isletlike clusters significantly produced insulin. The isletlike clusters could increase insulin secretion after a glucose challenge test.
Conclusions: This work provides a model for studying the differentiation of human-induced pluripotent stem cells to insulin-producing cells.
Key words : Pancreas, Islet cells, Induced pluripotent stem cells, Insulin
Autoimmune destruction of pancreatic endocrine beta cells leads to type 1 diabetes and the resistance against insulin affect results in type 2 diabetes mellitus. Current treatment for type 1 diabetes relies on insulin injection, and but this treatment often results in hypoglycemia and hyperglycemia. Even under controlled conditions, insulin therapy delays, but does not always prevent, development of complications.1 Complications such as ketoacidosis, kidney failure, heart disease, stroke, and blindness may develop in the patient because of uncontrolled hyperglycemia.2
Transplanting the entire pancreas may restore endogenous insulin production, but has the risk of perioperative morbidity because of damage from digestive enzymes from the exocrine pancreas during surgery.3-6 Regenerating insulin-producing beta cells and islet transplanting are long-term solutions for treating diabetes.1
In some patients, insulin independence with good glycemic control has been achieved and sustained for more than 2 years. Although promising, this approach faces multiple challenges. One is the shortage of donors compared with the large amount of patients. In addition, because of the low yield of islet cells from deceased-donor tissues, many donor cells are required to generate sufficient insulin-producing beta cells, which can produce and release adequate amounts of insulin in response to normal physiological signals.7-9
After a transplant, recurrence of the autoimmune response must be prevented to avoid destruction of donor cells. After an allograft transplant, chronic immunosuppression also is necessary to prevent rejection. These problems have caused many limitations that make this treatment difficult to be used by the general diabetic population. Therefore, deriving patient-specific isletlike cells from adult tissue may solve the shortage of organ donors and allograft rejection.10
Generating functional isletlike cell clusters (ILCs) from adult progenitor cells, including pancreatic progenitor cells, has been tested with limited success.11-13 Compared with adult progenitor cells, human embryonic stem cells (hESC) are a promising source for generating ILCs because of their unlimited replicative capacity and differentiation potentials.14
Transplanting pancreatic endoderm derived from hESC in vitro also has been shown to rescue mice with induced-insulin deficiency.15 However, applying human embryonic stem cell-derived ILCs was largely restricted by patient-specific embryonic stem cells.16-20
Reprogramming of somatic cells to pluripotent embryonic stem cell-like cells, termed induced pluripotent stem cells, has been performed with transcription factors, such as Oct4, Klf4, Sox2, c-Myc, and Lin28.16-19,21 Induced pluripotent stem cells have the same haplotype as the host; therefore, immuno-suppressive drug consumption to avoid rejection is not needed.22
This study was designed to generate ILCs using a human-induced pluripotent stem cell line. The differentiation was performed using a 4-step protocol over the course of 4 weeks. The findings suggest that it is possible to produce ILCs from human-induced pluripotent stem cells.
Materials and Methods
A human-induced pluripotent stem cell line (RSCB0082) was purchased from Iranian Royan stem cell bank.23 It had been produced by reprogramming a 42-year-old man’s dermal fibroblast cells. The induced pluripotent stem cells were grown on mitotically inactivated mouse embryonic fibroblasts in 70% Dulbecco's Modified Eagle Medium (DMEM)/F12 (Gibco [now Invitrogen Corporation], Carlsbad, CA, USA) supplemented with 20% knockout serum (Gibco), 0.1 mM nonessential amino acid (Gibco), 0.1 mM 2-mercaptoethanol (Sigma, St. Louis, MO, USA), insulin (5 mg/mL)-transferrin (5 mg/mL)-selenium (5 μg/mL) (Gibco), 100 IU/mL penicillin and 100 IU/mL streptomycin (Sigma) with 4 ng/mL basic fibroblast growth factor (Gibco). They were then incubated at 37°C with 5% CO2. All protocols were approved by the ethics committee of the institution before the study began, and conformed with the ethical guidelines of the 1975 Helsinki Declaration.
In vitro differentiation of pancreatic isletlike cell clusters
The 4-stage procedure for in vitro differentiation of induced pluripotent stem cells into ILCs was modified from a previously published protocol described by Chen and associates.24
For embryoid body formation, at first the iPSCs, colonies was trypsinized with trypsin-EDTA, and a single-cell suspension was made by pipetting the cells up and down. After counting the cells, we made hanging drops using 20 to 30 μL drops, containing 2000 cells, in a high DMEM media, 10% fetal bovine serum without bFGF, and they were placed on the lids of petri dishes, which then were incubated in CO2 for 3 days.
Three-day-old embryoid bodies were plated at a density of 100 embryoid bodies/well in 6-well culture plates. The tissue culture plates were coated with 0.1% gelatin and grown for 2 weeks in DMEM/F12 medium, 1:1, insulin (10 mg/L)-transferrin (6.7 ng/L)-a selenium (5.5 mg/L), (Gibco).
After 1 week, cells were separated with 0.05% trypsin-EDTA, and plated at a concentration of 2 × 105 per well in 6-well culture plates in DMEM/F12 media supplemented with 1% N-2 supplement, 2% B-27 supplement (Gibco), and 10 ng/mL fibroblast growth factor. Culture plates were coated with 0.1% gelatin (Sigma). It was during this stage that cell clusters formed.
Low DMEM supplemented with 1% N-2 and 2% B-27 was used at this stage. Then, fibroblast growth factor was removed, and 10 mM nicotinic acid (Sigma) was added.
Real-time qualitative polymerase chain reaction for pancreatic specific
transcription factor quantification
Total RNA was extracted from approximately 106 cells, using Mini-RNease - kit (CinnaGen, Tehran, Iran) according to the manufacturer’s instructions. The amount of extracted RNA was measured by OD260/280. cDNA was synthesized using M-MULV reverse transcriptase (CinnaGen) from 1 μg total RNAs to examine changes in the levels of pancreatic-specific transcription factors at the end of each stage, and they were normalized with beta-actin. All the experiments were performed in triplicate. Table 1 shows the real-time qualitative polymerase chain reaction (qPCR) primers, conditions, and product sizes. The cDNA was added to SYBR Green master mix (TaKaRa, Takara Shuzo, Otsu, Japan). Polymerase chain reaction amplifications were performed with StepOnePlus Real-Time PCR Systems (Applied Biosystems, Foster City, CA, USA).
β-actin was selected as an endogenous control, and the transcription of insulin, glucagon, PAX4, PAX6, NGN3, GLUT2, NKX6.1, KIR6.2, PDX 1, GLUT2, and OCT4 was checked in relation to β-actin. DNA amplification was performed under the following conditions:
The reaction included stage 1, initiating denaturation at 95°C for 5 minutes; stage 2, denaturation at 95°C for 1 minute; and annealing for 1 minute, for 40 cycles. A final stage was run to generate a melting curve to verify amplification product specificity.
At first, approximately 104 cells were fixed in acetone and placed on a slide for 3 minutes at 1000 rpm using a cytocentrifuge (Shafaara, Tehran, Iran). The peroxidase inactivation was performed by immersing it in 3% H2O2, and washing it in phosphate buffered saline for 5 minutes. During the last stage, cells were blocked with 1% blocking solution (1% bovine serum albumin and 0.1% Triton X-100 in phosphate buffered saline) (Dako A/S, Glostrup, Denmark) for 20 minutes. Slides then were incubated with anti-neurogenin3 antibody (abcam; Cambridge, MA, USA) (dilution 1:1000) and anti-Insulin (Dako A/S) (ready to use), for 40 minutes at room temperature. After washing in phosphate buffered saline, the Envision Detection System (Dako A/S) was added. The slides were washed with phosphate buffered saline for 10 minutes, visualized with diaminobenzidine (Dako A/S), and counterstained with hematoxylin (Sigma, USA). The cells in stage 1 served as negative controls.
Diphenylthiocarbazone, Dithizone (Sigma) is a zinc-binding chemical substance, and pancreatic islets containing zinc, stain red when treated by it. Cells in stages 2 and 3 served as negative controls for Dithizone staining.
Adherent clusters produced this way were rinsed twice in Krebs-Ringer HEPES buffer (125 mM NaCl, 4.74 mM KCl, 1 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 5 mM NaHCO3, 25 mM HEPES 1 mM, 0.1% bovine serum albumin, pH 7.4) containing 2.8 mM glucose (Sigma). Clusters then were incubated for 15 minutes, 30 minutes, 45 minutes, and 60 minutes in KRBH buffer with 16.7 mM (high-level) glucose. The insulin in the supernatant was determined by radioimmunoassay kit (Immunotech, Czech).
Data are presented as means ± SD. Each experiment was repeated 3 times. Data from the related assay were assessed by 1-way analysis of variance (ANOVA) followed by the Tukey test for pairwise comparison. To evaluate insulin secretion, study groups were compared using a 1-way ANOVA, followed the Tukey test. Values for P < .05 were taken to be statistically significant. The statistical analyses and design of the graphs were performed using GraphPad Prism 5 software (La Jolla, CA, USA).
Pluripotent stem cells were induced to differentiate into ILCs by the modified 4-stage protocol.
Morphologic changes in different stages
Induced pluripotent stem cells were differentiated to ILCs after 31 days (Figures 1A, 1B, 1C, and 1D).
Expression of pancreatic transcription factors in different stages
The gene expression pattern of differentiated cells at each stage were analyzed by real-time qPCR (Diagrams 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, and 1J).
Isletlike cell clusters and immunocytochemistry
During the fourth stage, ILCs were examined by immunocytochemistry for presence of insulin (Figure 2) and neurogenin 3 (Figure 3). More than 50% and 40% of the cells stained for insulin and neurogenin 3.
Isletlike cell clusters and Dithizone staining
From day 23, when the insulin-producing cells were formed, Dithizone was used to stain the cells, and the clusters became red (Figure 4).
Insulin secretion in isletlike cell clusters
As described in Materials and Methods section, after treating the ILCs with 16.7 mM glucose during the fourth stage, an increase in insulin secretion from 17.36 ± 11.112 pmol/L (2.5 ± 1.6 μIu/mL) to 2778 ± 141.81 pmol/L (400 ± 20.42 μIu/mL) was recorded. After incubating the cells for 5 to 60 minutes, the amount of secreted insulin in the medium was increased and maximum effect was seen after 60 minutes (Diagram 2).
Diabetes results from the loss or dysfunction of insulin-producing beta cells in the pancreas.25
The prevalence of type 1 and type 2 diabetes continues to increase. Pancreatic transplant has been the normal practice for treating diabetes. New investigations have focused on cell therapy for these patients. Cell replacement therapy through islet transplant is thought of as a potential long-term approach for controlling blood glucose levels. However, obtaining sufficient quantities of insulin-producing tissue for islet transplant remains an obstacle.5,6
Stem cells (either from embryonic stem cells or from pancreatic progenitors) could potentially provide an abundant alternate source of islet cells for transplant therapies.5,25 The promise of therapies derived from induced pluripotent stem, and stem cells, particularly holds high hopes for diabetes treatment.
During early pancreatic development, several transcription factors are activated, which ensure normal organogenesis and subsequent differentiation into endocrine cell types. Pancreatic organogenesis consists of a sequential cascade of inductive events along with the activation of specific transcription factors.26 Pdx1 is a transcription factor expressed during the early stages of pancreatic development, during islet cell differentiation, and in differentiation of beta cells.
Pdx1, PAX6, and NKX2-2 represent the core components of a transcription complex of islet-enriched genes that contribute to selectively regulating gene expression in beta cells during development.27 PAX4 expression is important for development of differentiating beta cells28 and contributes to maintaining NKX6-2 expression in differentiating beta cells.29 Neurogenin-3 (Ngn3), a basic helix-loop-helix transcription factor, functions as a pro-endocrine factor in the developing pancreas.30
Coexpression of insulin, Pdx1, and NKX6-1 is considered specific to mature beta cells.31 GLUT2, a glucose transporter with the lowest affinity and the highest capacity for glucose, is expressed in pancreatic beta cells. GLUT2 catalyzes glucose uptake by beta cells. This is the first step in signaling a cascade, leading to glucose-stimulated insulin secretion. GLUT2 is present in beta cells, but not in the other islet endocrine cells.32 KIR6.2 is a major subunit of the ATP-sensitive K+ channel and an inward-rectifier of potassium ion channels involved in insulin secretion.33 Homeodomain transcription factor Oct-4 is involved in the self-renewal of undifferentiated embryonic stem cells.34
We produced ILCs that had nearly the same characteristics as functional beta cells. The cells expressed beta-cell transcription factors including insulin, glucagon, PAX4, PAX6, NGN3, GLUT2, NKX6.2, KIR6.2, PDX1, and GLUT2. This gene expression profiling is similar to the key gene expression pattern of in vivo pancreas development. Immunocytochemistry also demonstrated that the NGN3 and insulin are expressed in ILCs; the percentage of NGN3 and insulin-expressing cells was approximately 40% and 50%. The ILCs secrete insulin after glucose stimulation and remained functional for approximately 5 months in vitro
The differentiation potential toward insulin-secreting cells has been seen in mouse studies,35-38 and hESC39 cells; but the existing differentiation protocols revealed limited efficiency, and the insulin-secreting level of these cells in vitro was lower than that of adult human islet cells.35-39
In 2011, Xu and associates studied treatment of human embryonic stem cells with fibroblast growth factor, activin A, and bone morphogenetic protein 4 for 3 to 4 days. Human embryonic stem cells treated with BMP4 expressed specific markers of pancreatic lineage and the foregut of endodermal cells and more differentiation after using ITS, GF7, and nicotinamide.40
In 2012, Liu and associates offered a 2-step protocol in 7 days for the differentiation of mouse embryonic stem cells into insulin-producing cells using activin A in the first step and nicotinamide, insulin, and laminin in the second.41
In 2012, Cheng and associates showed that endodermal progenitor cell lines derived from induced pluripotent and human embryonic stem cells could differentiate into numerous endodermal lineages, such as monohormonal glucose-responsive pancreatic β cells, hepatocytes, and intestinal epithelia, by manipulation of in vitro culture.42 In 2009, Zhang and associates reported an approach to induce human embryonic stem cells and pluripotent stem cells to differentiate into ILCs in 22 days in a chemical-defined culture system including N2, B27, activin A, retinoic acid, FGF7, NOGGIN, EGF, fibroblast growth factor, Exendin-4, and BMP4. They confirmed that epidermal growth factor facilitated expansion of PDX1-positive pancreatic progenitors.43 The differentiated cells obtained by this method comprised nearly 25% insulin positive cells as assayed by flow cytometry analysis. The previously reported efficiency for insulin/C-peptide–positive cells as assayed by flow cytometry analysis was 4.1% or 7.3%.14,44,45 In 2010, Alipio and associates differentiated induced pluripotent stem cells from mice to betalike cells by using laminin, insulin, nicotinamide, selenic acid, transferrin, and progesterone.46
Thatava and associates generated patient-specific pluripotent stem cells from a diabetic man by nonintegrating Sendai viral vectors, and then differentiated the cells into functional insulin-producing cells. The authors believe that this method allows reproducible generation of genomic modification-free induced pluripotent stem cells from diabetic patients for autologous cell replacement therapy.47 The virus-mediated delivery of reprogramming factors, such as c-Myc and KLF4, results in permanent integration of these oncogenes with subsequent permanent genetic alterations.48-49 Therefore, various methods have been used to generate induced pluripotent stem cells with lower genetic integration such as mRNAs, plasmid transient transfection, episomal vector, nonintegrating Sendi virus, and adenovirus. Induced pluripotent stem cells usually are grown on feeder cells, such as primary mouse embryonic fibroblasts or mouse embryonic fibroblast cell line, in media-containing fetal calf serum. These conditions expose the patients to animal pathogens or lead to immune rejection.48-49
Although we produced ILCs in a chemical-defined culture system, our culture medium was not xeno-free, and the genomic modification in induced pluripotent stem cells was done by integrating the retroviral vectors. Therefore, the important issues that must be addressed are as follows:
(1) Substantial efforts must be performed to produce functional ILCs in
vector-free and xeno-free environment.
(2) The engraftment efficacy of these cells must be evaluated in an animal model.
(3) Does transplant of these induced pluripotent stem cells derived from ILCs secrete insulin physiologically, and are they effective in reducing the hyperglycemic phenotype of animal models?
(4) Does immuno-rejection of the transplanted cells occur?
(5) Do these cell clusters continue to grow and develop into a tumor and metastasize in vivo?
Volume : 13
Issue : 1
Pages : 68 - 75
DOI : 10.6002/ect.2013.0131
From the 1Department of Biology, Science and Research Branch,
Islamic Azad University, Fars; the 2Transplant Research Center,
Shiraz University of Medical Science, Shiraz, Iran; and the 3Department
of Biology, Kazerun Branch, Islamic Azad University, Kazerun, Iran
Acknowledgements: Anahita Shaer, Negar Azarpira, and Mohammad Hosein Karimi participated in the study design. Anahita Shaer did all the experiments, gathered the data, and performed the statistical analyses. All of the authors participated in the manuscript preparation. Anahita Shaer wrote the manuscript, with valuable assistance from Negar Azarpira.
We would like to thank the Research Improvement Center of Shiraz University of Medical Sciences, Shiraz, Iran, and Ms. A. Keivanshekouh for improving the English in the manuscript. We also are grateful to Mrs. Elaheh Esfandiari and Mrs. Masoumeh Darai for assisting with the cell cultures and polymerase chain reaction.
The authors declare that they have no financial interests related to the material in the manuscript. The study was financially supported by a grant from Iran National Science Foundation (INSF). The authors have no conflicts of interest to declare.
Corresponding author: Anahita Shaer, Department of Biology, Science and Research Branch, Islamic Azad University, Fars, Iran
Phone: +987 11 647 4331
Fax: +987 11 647 4331
Table 1. Real-Time Qualitative Polymerase Chain Reaction Primer Sequences and Product Sizes
Figure 1. In Vitro Differentiation Scheme for Generating ILCs
Diagram 1. Real-Time Qualitative Polymerase Chain Reaction Determination of Gene Expression in Different Differentiation Stages
Figure 2. Insulin-Producing Cells Stain Positive for Insulin Expression (×100)
Figure 3. Insulin-Producing Clusters Stain Positive for Neurogenin 3 (×100)
Figure 4. Selective Dithizone Staining of ILCs (Red Color) (×100)
Diagram 2. Insulin Release in Response to 16.7 mM Glucose in Stage 4