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
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
Conclusions: This work provides a model for studying the
differentiation of human-induced pluripotent stem cells to insulin-producing
Key words: Pancreas, Islet cells, Induced pluripotent stem cells,
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
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
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
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
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
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
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
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
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
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
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
(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?
- Efrat S. Beta-cell replacement for insulin-dependent diabetes mellitus.
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- Atkinson MA, Eisenbarth GS. Type 1 diabetes: new perspectives on disease
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Volume : 13
Issue : 1
Pages : 68-75
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
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
Figure 3. Insulin-Producing Clusters Stain Positive for Neurogenin 3
Figure 4. Selective Dithizone Staining of ILCs (Red Color) (×100)
Diagram 2. Insulin Release in Response to 16.7 mM Glucose in Stage 4