Maehr, R. et al. Generation of pluripotent stem cells from patients with type 1 diabetes. Proc. Natl Acad. Sci. USA 106, 15768-15773

Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 09/2009; 106(37):15768-73. DOI: 10.1073/pnas.0906894106
Source: PubMed
Type 1 diabetes (T1D) is the result of an autoimmune destruction of pancreatic beta cells. The cellular and molecular defects that cause the disease remain unknown. Pluripotent cells generated from patients with T1D would be useful for disease modeling. We show here that induced pluripotent stem (iPS) cells can be generated from patients with T1D by reprogramming their adult fibroblasts with three transcription factors (OCT4, SOX2, KLF4). T1D-specific iPS cells, termed DiPS cells, have the hallmarks of pluripotency and can be differentiated into insulin-producing cells. These results are a step toward using DiPS cells in T1D disease modeling, as well as for cell replacement therapy.


Available from: René Maehr, Nov 17, 2014
Generation of pluripotent stem cells from patients
with type 1 diabetes
, Shuibing Chen
, Melinda Snitow
, Thomas Ludwig
, Lisa Yagasaki
, Robin Goland
, Rudolph L. Leibel
and Douglas A. Melton
Department of Stem Cell and Regenerative Biology, Howard Hughes Medical Institute, Harvard Stem Cell Institute, Harvard University, 7 Divinity Avenue,
Cambridge, MA 02138; and
Department of Pathology and Cell Biology, and
Division of Molecular Genetics and Naomi Barrie Diabetes Center, College
of Physicians and Surgeons, Columbia University, New York, NY 10032
Contributed by Douglas A. Melton, July 8, 2009 (sent for review May 18, 2009)
Type 1 diabetes (T1D) is the result of an autoimmune destruction
of pancreatic
cells. The cellular and molecular defects that cause
the disease remain unknown. Pluripotent cells generated from
patients with T1D would be useful for disease modeling. We show
here that induced pluripotent stem (iPS) cells can be generated
from patients with T1D by reprogramming their adult fibroblasts
with three transcription factors (OCT4, SOX2, KLF4). T1D-specific
iPS cells, termed DiPS cells, have the hallmarks of pluripotency and
can be differentiated into insulin-producing cells. These results are
a step toward using DiPS cells in T1D disease modeling, as well as
for cell replacement therapy.
cell disease model autoimmune directed differentiation endoderm
he study of human disease is of ten hindered by the lack of a
good model system. The initiation of the primary disease
process often occurs long before the patient shows any sign of
disease. Also, relevant patient tissue can be limited and difficult
to obtain. Although rodent models can give valuable insights,
these rarely fully recapitulate the human disease. Although the
nonobese diabetic (NOD) mouse has been enormously useful,
there are justifiable concerns regarding its validity as a model for
human type 1 diabetes (T1D) (1–3). Recently, human induced
pluripotent stem (iPS) cells with disease genot ypes have been
generated as a tool for human disease modeling (4–7). Disease-
relevant cell t ypes can be generated via in v itro differentiation
protoc ols, and in the best cases, these protoc ols enable an in vitro
analysis of the disease pathology. Although ES cells are the gold
st andard for pluripotent stem (PS) cells, ES cells only model
diseases that can be diagnosed or predicted by simple Mendelian
genetics [e.g., cystic fibrosis (8) and Fanconi Anemia (9)]. Our
focus lies in understanding T1D, a disease with complex under-
lying genetics and unidentified environmental triggers. For T1D,
as well as other multigenic diseases, iPS cells are the best starting
point, because they are derived from patient cells and, thereby,
capture the disease genotype in a stem cell. To this date, it is not
clear whether different forms of t ype 1 diabetes exist, and how
genetics and environment factor influence each other in disease
onset and prog ression.
T1D results from the destruction of insulin-producing
by the body’s own immune system. A cure could be achieved by
c ombining
cell replacement therapy with induction of toler-
ance to such cells. Cell replacement therapy for T1D requires a
source of glucose-responsive, insulin-secreting cells. Promising
results have been obtained by transplantation of pancreatic islets
of Langerhans or pancreatic tissue, but this approach is circum-
scribed by the limited and irregular supply of cadaveric donor
tissue, as well as the risks of treatment with immunosuppressant
dr ugs (10, 11). An alternative source of insulin-producing cells
are PS cells that can be differentiated into pancreatic
cells. For
example, ES cells have been differentiated in monolayer culture
along the endoder mal lineage toward insulin-producing cells
(12, 13). However,
cells derived from immunologically un-
matched ES cells will likely be the targets of both allograft
reactions and the autoimmune response that caused the initial
cell destruction.
Mouse and human fibroblasts can be used to generate iPS cells
(14–16). Recently, iPS cells have been generated from fibro-
blasts obtained from patients with various diseases (4–7), but not
for T1D. T1D-specific iPS (DiPS) cells derived from patients
of fer several significant advantages. First, DiPS cells would
unquestionably contain the genotype responsible for the human
disease. Second, DiPS cells would provide an immunologically
matched autologous cell population, although dependent on
improvements in dif ferentiation protocols. Third, and the
present focus of our work, patient-specific cells make possible
patient-specific disease modeling wherein the initiation and
progression of this poorly understood disease can be studied.
Because DiPS cells can be manipulated and studied in vitro, one
should be able to assess how the different cell types, including
dif ferentiated
cells, and immunocytes interact to produce a
pathological phenotype. The purpose of the present study was to
derive DiPS cells f rom patients with T1D, and to test whether
these cells can be differentiated into the major target cell type,
the pancreatic
cell. Extending this approach to all cell t ypes
involved in T1D could lead to an understanding of the root
causes of the disease and to the development of effective
prophylactic and therapeutic strategies.
Generation of iPS Cells from Patients with T1D. Skin biopsies were
obt ained from two Caucasian males with T1D of 11- and
27-years duration, respectively. Patient 1 was presented at age 21
with polyuria, polydypsia, and a blood glucose concentration of
680 mg/dL. Patient 2 was presented at age 3 in diabetic ketoac-
idosis requiring hospitalization. For both indiv iduals, the body
mass index did not exceed 22 or 23, respectively. HLA haplo-
t ypes, and other clinical data, are given in Table 1. Fibroblasts
obt ained from skin biopsies were cultured and infected with a
c ombination of retroviruses encoding the transcription factors
OCT4, SOX2, and K LF4 (15). Starting 4 weeks after infections,
c olonies were picked based on their morphological resemblance
to human ES cell c olonies and expanded. To test whether the cell
lines expressed pluripotency markers, we verified the presence of
alk aline phosphatase (AP) activity, as well as staining for
antibodies to OCT4, NANOG, SOX2, TRA1-60, TRA1-81, and
SSEA4. The reprogrammed cells were positive for AP activ ity,
and were reactive to antibodies against all pluripotency markers
Author contributions: R.M., R.G., R.L.L., and D.A.M. designed research; R.M., S.C., M.S., and
L.Y. performed research; T.L.,R.G., and R.L.L. contributed new reagents/analytic tools; R.M.,
S.C., L.Y., R.L.L., and D.A.M. analyzed data; and R.M. and D.A.M. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
See Commentary on page 15523.
To whom correspondence should be addressed. E-mail:
This article contains supporting information online at
September 15, 2009
vol. 106
no. 37 www.pnas.orgcgidoi10.1073pnas.0906894106
Page 1
tested (Fig. 1 A and B). Karyot ype analysis and DNA finger-
printing verified that the T1D patient-specific DiPS lines were
generated f rom the parent al fibroblast line and maintained a
nor mal karyotype (Fig. S1 and Table S1).
The analysis of pluripotency markers was extended to include
ex pression of the endogenous genes encoding NANOG, OCT4,
TERT, REX1, SOX2, and GDF3 by semiquantitative PCR
analysis. Expression of these genes in the DiPS lines was similar
to human ES cells, but was absent in the parental fibroblasts (Fig.
2A). As previously described, KLF4 is ex pressed in fibroblasts,
as well as in human ES cells and DiPS cells (Fig. 2 A) (15).
Ex pression of
-ACTIN was used as a control for RNA recovery
and to allow semiquantitative comparison of expression levels.
Omission of the reverse transcription reaction was used as a
c ontrol for specificity and gave no bands of expected size in the
semiquantit ative PCRs (Fig. 2 A). A more global view of the gene
ex pression profiles for the parental fibroblasts, DiPS, and hES
lines, was obtained using DNA microarrays. Hierarchical clus-
tering revealed that DiPS cell lines from both patients were
highly similar to the human ES cell lines HUES4, HUES6, and
HUES8 (17), while exhibiting low similarity to fibroblasts (Fig.
2B). The coefficient of determination (r
square of the
c orrelation coefficient) was 0.72–0.74 for DiPS compared with
parent al fibroblasts, and 0.940.98 for DiPS compared with
HUES cells (Table S2). We conclude that DiPS cells closely
resemble human ES cells in global gene expression as we have
described for iPS from primary human cells before (18).
To determine whether the viral transgenes were silenced, we
performed exogenous gene-specific quantitative PCR analysis.
Compared with the infected fibroblast control, the transgene
ex pression in the DiPS cells was low or at background levels (Fig.
2C), presumably due to anticipated viral gene silencing (15). We
c onclude that the DiPS lines closely resemble human ES cells in
their ex pression profiles and, like other reported iPS cells, have
silenced the transgenes.
DiPS Cells Spontaneously Differentiate into Cell Types of Different
Germ Layers.
DiPS cells were allowed to spontaneously differen-
tiate in embryoid body (EB) cultures. EB for mation was
achieved by culturing DiPS cells in differentiation media on
low-att achment plates, followed by plating onto gelatin-coated
dishes for additional culture. We analyzed the morphologically
dif ferentiated cells for expression of markers for the endodermal
(SOX17 and FOXA2), mesodermal (SMA), and ectoder mal
(T UJ1) lineages in differentiated cultures (Fig. 3A). The differ-
entiated DiPS cells were found positive for cells of all three germ
layers. Also, we verified pluripotency of DiPS cells in teratoma
for mation assays. After injection of DiPS cells into immuno-
c ompromised mice, DiPS cells for med teratomas containing
derivatives of endoderm (glandular str uctures), mesoderm (car-
tilage), and ectoder m (nerve fibers, pigmented epithelium, and
melanoc ytes) (Fig. 3B). We conclude that patient-specific DiPS
cells can spontaneously differentiate into derivatives of all three
ger m layers.
DiPS Cells Can Be Differentiated Along the Endodermal/Pancreatic
We applied a directed differentiation protocol to the
DiPS cells to determine whether they can be differentiated
toward an insulin producing/glucose responsive cell. The proto-
c ol followed a stepwise dif ferentiation protocol that relies on the
Table 1. Patient information
Patient Race Age/sex
at age of
History of
index Medication
A1C, % HLA
H1 Caucasian 32/male 21 First cousin with T1D Yes 22 Insulin 45 U/day sub. cut. 6.6 DR1/4
H2 Caucasian 30/male 3 None Yes 23 Insulin 60 U/day sub. cut. 7.2 DR1/13
Fig. 1. Generation of DiPS cells from T1D patients. DiPS lines were established from two T1D affected patient fibroblasts lines H1 (A) and H2 (B). Displayed are
DiPS lines (A) H1.5 and (B) H2.4. Detection of AP activity and immunofluorescence analyses for presence of pluripotency markers OCT4, SSEA4, NANOG, TRA1-60,
SOX2, and TRA1-81 are indicated. For immunofluorescence stains corresponding nuclear stains (DAPI) visualize all cells including mouse embryonic fibroblast
feeder cells.
Maehr et al. PNAS
September 15, 2009
vol. 106
no. 37
Page 2
generation of intermediate precursors thought to be similar to
populations present in the developing embryo. DiPS cells were
subjected to a protocol that directs differentiation to definitive
endoder m followed by gut tube endoderm before further dif-
ferentiation into pancreatic progenitors, and finally, the
cell (Fig. 4A). As determined by immunofluorescence analysis
for SOX17 and FOXA2 expression, DiPS lines from both
patients could respond to WNT3A and Activin A treatment to
dif ferentiate into definitive endoder m, similar to human ES cells
(Fig. 4B Upper). Further dif ferentiation toward gut tube
endoder m (HNF4a and HNF1b positive), and pancreatic pro-
gen itors (PDX1 and HNF6 positive) was achieved by supplying
FGF10 and cyclopamine, and FGF10, cyclopamine, retinoic
acid, and ()-Indolactam V, respectively. FOXA2 expression
was detected in both definitive endoderm and pancreatic pro-
gen itors (Fig. 4B) as expected from normal embryonic devel-
opment (19). The expression of these transcription factors was
validated by semiquantitative PCR (Fig. 4C).
Further differentiation of DiPS-derived pancreatic progeni-
tors toward the endocrine lineage yielded cells that were positive
for somatostatin, glucagon, and insulin (Fig. 5A). Insulin-positive
cells were C-peptide positive, excluding insulin uptake from the
media. Semiquantitative PCR analysis confirmed the expression
of endocrine-specific gene products of INSULIN, PDX1,
address whether the insulin could be released on gluc ose stim-
ulation in vitro, we exposed the DiPS-derived insulin producing
cells to low or high concentrations of glucose. The DiPS-derived
population released human C-peptide on glucose stimulation
(Fig. 5C). The amount of released C-peptide after high (20 mM)
gluc ose stimulation was at least 5-fold higher than after low (2.5
mM) glucose stimulation. We c onclude that DiPS cells can be
dif ferentiated to insulin producing/glucose-responsive cells.
Insight into the pathogenesis of T1D comes largely from rodent
models such as the NOD mouse or the BB rat. However, the
existing rodent models are not fully repre sentative of the relevant
processe s in patients with T1D. Indeed, insights from rodent models
have frequently not translated well to the clinic (1–3). Access to
genetically-predisposed human cells whose biological status ulti-
mately defines the disease enables previously impossible mecha-
nistic studies in vitro, and in vivo transplant systems. We have
generated human PS cells from two patients with T1D. These DiPS
cells provide a starting material for patient-specific disease mod-
eling and for testing differentiation protocols. In mice, inclusion of
c-Myc as a fourth reprogramming factor is associated with lethal
tumor formation in contrast to reprogramming with three factors
(Oct4, Sox2, and Klf4) (20). The reprogramming process described
here was achieved by using three factors (OCT4, SOX2, and KLF4),
omitting the oncogene C-MYC. Consistent with previously de-
scribed iPS cell lines, transgene silencing was observed in DiPS cells
(4, 15, 21, 22). Induced PS cells can be generated from mouse cells
without permanent or transient integration of the transforming
factors in the genome (23, 24), and this approach has recently
extended to human cells (25). Currently, generation of human iPS
cells involve s delivery of DNA in a manner that allows potential
integration into the genome, but alternative approache s are likely
to be available in the near future (26). In any event, insufficient
characterization of the reprogramming process and its product
precludes use of iPS cells in cell replacement therapy at this point.
However, the generation of patient-specific PS cells paired with
differentiation into cell types relevant to the disease promises to
provide valuable insights into disease pathogenesis. The DiPS cells
described here are pluripotent based on similarities in gene expres-
sion to human ES cells and their ability to spontaneously differ-
entiate into cells of different germ layers. Notably, we have differ-
entiated these patient-specific DiPS cell lines to a cell population
relevant to T1D, an insulin-producing and glucose-responsive
-like cell. Previously, iPS cells generated from foreskin fibroblasts,
but not from diabetes patients, using four factor reprogramming
(OCT4, SOX2, KLF4, and C-MYC) were shown to differentiate to
insulin-producing clusters (27), leaving open the task of generating
patient-specific DiPS, and testing their potential to differentiate
toward insulin-producing
-like cells. Although DiPS cells can be
differentiated to insulin producing cells, the efficiency of this
process is low, possibly because the differentiation protocol has not
been optimized or due to variation in the differentiation propen-
sities of human PS cells (28). Interestingly, we observed differences
between DiPS lines from the same patient, potentially due to
transgene reactivation or incomplete silencing. As a consequence,
characterization of multiple lines and future efforts to generate
DiPS cells without viral integration will help address this issue.
As observed with human ES cells,
-like cells derived from
DiPS are glucose responsive, but until the differentiation pro-
toc ols are improved, it is not yet possible to directly compare
these cells with purified pancreatic
cells. In addition to
Fig. 2. Expression analysis of patient specific DiPS cells. (A) Semiquantitative
analysis of expression of OCT4, SOX2, REX1, NANOG, KLF4, GDF3, and
Control PCR (no RT) is included. (B) Hierarchical cluster analysis of different
DiPS, HUES and fibroblast lines. (C) Quantitative assessment of viral transgene
expression (tgOCT4, tgSOX2, and tgKLF4) levels. Viral transgene expression
was normalized to control infected BJ fibroblasts (isolation occurred 7 days
post infection). Uninfected HUES and fibroblast lines were used as controls.
The experiment was performed in duplicates and the error bars represent SD.
www.pnas.orgcgidoi10.1073pnas.0906894106 Maehr et al.
Page 3
variation in differentiation propensities, potential deviations in
T1D disease onset and progression will require the generation of
multiple DiPS lines to reflect the human population afflicted
with T1D. The two cell lines from different patients described
here represent a starting point for this larger t ask.
Dif ferentiation of DiPS cells to
-like cells is relevant not only
for the long ter m possibility of autologous cell replacement
therapy, but also for disease modeling. We propose to extend this
investigation to generate cell t ypes of the immune system that
may allow the generation of a cellular interaction model of T1D.
This approach should provide a way to investigate T1D disease
onset and progression in vitro and/or in reconstituted animal
models. These in vitro and in vivo systems will also be useful for
testing of preventative and therapeutic strategies.
Materials and Methods
Cell Culture. Skin fibroblasts from T1D patients and controls were derived from
explants of 3-mm dermal biopsies after informed consent under protocols
approved both by Harvard University and Columbia University College of
Physicians and Surgeons. Briefly, 3-mm skin biopsies were minced with scalpels
into smaller pieces, and tissue fragments were placed into a 60-mm tissue
culture dish under a sterile coverslip held down by sterilized silicon grease
under one corner. Media was added to completely immerse the coverslip, and
dishes were incubated at 37 °C in a humidified incubator (5% CO
). Media was
changed every 5 days without disturbing the coverslip. Fibroblasts grew out of
the tissue fragments, and when sufficiently numerous, cells were trypsinized
and expanded. Subsequently, fibroblasts were maintained in fibroblast me-
dium (DMEM supplemented with 10% FBS, glutamine, sodium pyruvate,
nonessential amino acids, and penicillin/streptomycin). The resulting fibro-
blasts lines are referred to as fibroblast Harvard (H) lines 1 and 2.
Human ES cell and DiPS lines were cultured in human ES media (knockout
DMEM supplemented with 10% knockout serum replacement, 10% human
plasma fraction, 10 ng/mL bFGF, nonessential amino acids,
nol, L-glutamine, and penicillin/streptomycin). Cultures were maintained on
mouse embryonic fibroblast feeders and passaged enzymatically using either
0.05% Trypsin (GIBCO) or Collagenase type IV.
Fig. 3. Spontaneous differentiation of DiPS cells into cells of different germ layer origin. (A) In vitro differentiation of DiPS lines H1.5, H2.1, and H2.4 in EB assays
was followed by monolayer culture and immunostaining for markers of ectoderm (TUJ1), mesoderm (SMA), and endoderm (FOXA2 and SOX17). An overlay with
a nuclear stain (DAPI) is displayed. (B) Teratoma formation occurred after injection of DiPS into immunocompromised mice. Hematoxylin and Eosin staining of
teratoma sections shows nerve fibers (N), melanocytes (M), pigmented epithelium (P), cartilage (C), and glandular structures (G).
Fig. 4. Stepwise differentiation of ES/DiPS cells toward
-like cells. (A) Schematic representation of stepwise differentiation of human PS cells to
-like cells.
DiPS cell lines H1.5, H2.1, and H2.4 differentiation to definitive endoderm (DE), gut tube endoderm (GTE) and pancreatic progenitors (PPs) indicated by (B)
immunostaining and (C) RT-PCR. SOX, SRY (sex determining region Y)-box; FOXA2, forkhead box protein A2; HNF, hepatocyte nuclear factor; PDX1, pancreatic
and duodenal homeobox 1; HB9, homeobox gene HLXB9; NKX6.1, NK6 transcription factor related, locus 1.
Maehr et al. PNAS
September 15, 2009
vol. 106
no. 37
Page 4
Reprogramming. VSVG-coated retroviruses were generated according to stan-
dard procedures. One day before infection, 10E5 fibroblasts were seeded per
well of a six well plate. Fibroblasts were infected on days 1 and 2 with a
combination of OCT4, SOX2, and KLF4 containing Moloney viruses (constructs
were obtained from Addgene) (15). The media was changed on day 3 to
DMEM supplemented with 10% FBS, L-glutamine, penicillin/streptomycin,
nonessential amino acids, and sodium pyruvate. A day later the cells were split
onto gelatinized 10-cm cell culture dishes. Subsequently, the cells were fed
every other day with human ES cell media. Generation of DiPS lines H2.4, H2.3,
and H2.1b occurred with supplementation of the media with 1 mM valproic
acid during the reprogramming process as described before (18), whereas DiPS
lines H2.1, H1.1, H1.5 were generated without addition of the chemical.
Colonies were picked starting 4 weeks after infection.
Spontaneous Differentiation. Spontaneous differentiation through EB forma-
tion was initiated by dissociation of human DiPS cells using collagenase IV
treatment, and subsequent transfer to low attachment 6-well plates in knock-
out DMEM supplemented with 20% knockout serum replacement, nonessen-
tial amino acid,
-mercaptoethanol, L-glutamine, and penicillin/streptomycin.
After 8 –10 days suspension culture, EBs were transferred to gelatin-coated
plates and cultured for an additional 8–10 days as attachment culture.
For teratoma formation assays DiPS cells were collected by collagenase IV
treatment, and injected s.c. into immunocompromised mice (NOD-SCID or
SCID-Beige mice). Teratomas were collected 7–10 weeks after injection, and
processed according to standard procedures for paraffin embedding and
hematoxylin and eosin staining.
Directed Differentiation. Directed differentiation was conducted as described
(13, 29) with the following modifications: human DiPS cells were cultured on
MEF feeder cells to 70 80% confluency, then treated with 25 ng/mL WNT3A
(R&D systems) 100 ng/mL Activin A (R&D systems) in advanced RPMI (A-RPMI;
Invitrogen) supplemented with 1L-Glu and 1PS for 1 day, followed by
treatment with 100 ng/mL Activin A in A-RPMI supplemented with 1L-Glu,
1PS and 0.2% FBS (Invitrogen). Two days later, the media was changed to 50
ng/mL FGF10 (R&D systems) 0.25
M KAAD-CYC (Calbiochem) in A-RPMI
supplemented with 1L-Glu, 1PS and 2% FBS and maintained for additional
2 days. Cells were then transferred to 50 ng/mL FGF10 0.25
M RA (Sigma) in DMEM supplemented with 1L-Glu, 1PS, 1 B27 (Invitro-
gen) and cultured for an additional 4 days. The media was then changed to 50
ng/mL FGF10 300 nM ILV (Axxora) in DMEM supplemented with 1L-Glu,
1PS, 1 B27 and cultured for an additional 4 days. Then, cells were trans-
ferred to 50 ng/mL EX-4 (Sigma) 10
M DAPT (Sigma) in DMEM supple-
mented with 1L-Glu, 1PS, 1 B27 and cultured for an additional 6 days.
Cells were then cultured in 50 ng/mL HGF (R&D systems) 50 ng/mL IGF1 (R&D
systems) in CMRL-1066 (Invitrogen) supplemented with 1L-Glu, 1PS, 1
B27 for 6 days.
Immunofluorescence. Immunofluorescence staining was performed using
primary antibodies against C-peptide (4020-01; Linco), FOXA2 (07-633;
Upstate), glucacon (4031; Linco), HNF6 (sc-13050; Santa Cruz Biotechnol-
ogy), insulin (A0564; Dako), NANOG (ab21624; Abcam), NKX2.5 (sc-14033;
Santa Cruz Biotechnology), OCT4 (sc-5279; Santa Cruz Biotechnology),
PDX1 (AF2419; R&D systems), SMA (A5228; Sigma), somatostatin (A0566;
Dako), SOX2 (sc-17320; Santa Cruz Biotechnology), SOX17 (AF1924; R&D
systems), SSEA4 (MAB4304; Chemicon), TRA-1– 60 (MAB4360; Chemicon),
TRA-1–81 (MAB4381; Chemicon), and TUJ-1 (MMS-435P; Covance Research
Products). Appropriate secondary antibodies were obtained from Molec-
ular Probes.
Gene Expression Analysis. RNA was isolated from cells using RNAeasy kit
(Qiagen). For quantitative and semiquantitative PCR analysis, cDNA synthesis
was performed using SuperScript III Reverse Transcriptase and Oligo (dT)
primers (Invitrogen). Primers used for amplification are listed in Table S3.
For whole-genome expression analysis, Illumina Total Prep RNA amplification
Kit (Ambion) was used according to manufacturer’s guideline. Hybridization to
Whole-Genome Expression BeadChips (HumanRef-8) was followed by analysis on
an Illumina Beadstation 500. All samples were prepared in duplicates. Data
analysis was conducted using manufacturer’s Beadstudio software.
C-Peptide Release Assay. C-peptide release was measured by incubating the
cells in Krebs–Ringer solution containing bicarbonate and Hepes (KRBH; 129
Fig. 5. DiPS cell lines H1.5, H2.1, and H2.4 differentiate to hormone-expressing endocrine cells indicated by (A) immunostaining and (B) semiquantitative PCR.
(C) The DiPS-derived C-peptide-expressing cells secreted C-peptide on glucose stimulation. The DiPS-derived populations were stimulated with 2.5 and 20 mM
D-glucose, and the amount of human C-peptide released to culture supernatant was analyzed by ELISA. C-PEP, C-peptide; INS, insulin; GLU, glucagon; SS,
www.pnas.orgcgidoi10.1073pnas.0906894106 Maehr et al.
Page 5
mM NaCl/4.8 mM KCl/2.5 mM CaCl
/1.2 mM KH
/1.2 mM MgSO
/5 mM
/10 mM Hepes/0.1% BSA). The cells were incubated in KRBH buffer for
1 h to wash. The cells were incubated in KRBH buffer with 2.5 mM D-glucose
for 1 h and then KRBH buffer with 20 mM D-glucose for 1 h. The C-peptide
levels in culture supernatants were measured using the human C-peptide
ELISA kit (Alpco Diagnostics).
ACKNOWLEDGMENTS. We thank Kevin Eggan for organizational help and
discussions, Anastasie Kweudjeu for help with transcriptional arrays, Danwei
Huangfu for helpful discussions and experimental advice, Mariko Yamaki and
Adriana Tajonar for technical help, Julian McKay-Wiggan, M.D. for perform-
ing skin biopsies, and Taylor Armstron and Sunanda Babu (University of
Colorado, Aurora, CO) for islet antibody and HLA typing. S.C. is supported by
the postdoctoral fellowship from Juvenile Diabetes Research Foundation.
D.A.M. is an Investigator of the Howard Hughes Medical Institute. This work
was supported by the Harvard Stem Cell Institute (Kurtzig Fund), the Russell
Berrie Foundation, the Handler Foundation, and the New York Stem Cell
1. Roep BO (2007) Are insights gained from NOD mice sufficient to guide clinical trans-
lation? Another inconvenient truth. Ann N Y Acad Sci 1103:1–10.
2. Roep BO, Atkinson M (2004) Animal models have little to teach us about type 1
diabetes: 1. In support of this proposal. Diabetologia 47:1650 –1656.
3. von Herrath M, Nepom GT (2009) Animal models of human type 1 diabetes. Nat
Immunol 10:129–132.
4. Dimos JT, et al. (2008) Induced Pluripotent Stem CellsGenerated from Patients with ALS
Can Be Differentiated into Motor Neurons. Science 321:1218 –1221.
5. Ebert AD, et al. (2009) Induced pluripotent stem cells from a spinal muscular atrophy
patient. Nature 457:277–280.
6. Park IH, et al. (2008) Disease-Specific Induced Pluripotent Stem Cells. Cell 134:877–886.
7. Soldner F, et al. (2009) Parkinson’s disease patient-derived induced pluripotent stem
cells free of viral reprogramming factors. Cell 136:964 –977.
8. Pickering SJ, et al. (2005) Generation of a human embryonic stem cell line encoding the
cystic fibrosis mutation deltaF508, using preimplantation genetic diagnosis. Reprod
Biomed Online 10:390 –397.
9. Verlinsky Y, et al. (2005) Human embryonic stem cell lines with genetic disorders.
Reprod Biomed Online 10:105–110.
10. Naftanel MA, Harlan DM (2004) Pancreatic islet transplantation. PLoS Med 1:e58; quiz
11. Lakey JR, Mirbolooki M, Shapiro AM (2006) Current status of clinical islet cell trans-
plantation. Methods Mol Biol 333:47–104.
12. D’Amour KA, et al. (2006) Production of pancreatic hormone-expressing endocrine
cells from human embryonic stem cells. Nat Biotechnol 24:1392–1401.
13. Kroon E, et al. (2008) Pancreatic endoderm derived from human embryonic stem cells
generates glucose-responsive insulin-secreting cells in vivo. Nat Biotechnol 26:443–
14. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse
embryonic and adult fibroblast cultures by defined factors. Cell 126:663– 676.
15. Takahashi K, et al. (2007) Induction of pluripotent stem cells from adult human
fibroblasts by defined factors. Cell 131:861– 872.
16. Yu J, et al. (2007) Induced pluripotent stem cell lines derived from human somatic cells.
Science 318:1917–1920.
17. Cowan CA, et al. (2004) Derivation of embryonic stem-cell lines from human blasto-
cysts. N Engl J Med 350:1353–1356.
18. Huangfu D, et al. (2008) Induction of pluripotent stem cells from primary human
fibroblasts with only Oct4 and Sox2. Nat Biotechnol 26:1269 –1275.
19. Monaghan AP, Kaestner KH, Grau E, Schutz G (1993) Postimplantation expression
patterns indicate a role for the mouse forkhead/HNF-3 alpha, beta and gamma genes
in determination of the definitive endoderm, chordamesoderm and neuroectoderm.
Development 119:567–578.
20. Nakagawa M, et al. (2008) Generation of induced pluripotent stem cells without Myc
from mouse and human fibroblasts. Nat Biotechnol 26:101–106.
21. Lowry WE, et al. (2008) Generation of human induced pluripotent stem cells from
dermal fibroblasts. Proc Natl Acad Sci USA 105:2883–2888.
22. Park IH, et al. (2008) Reprogramming of human somatic cells to pluripotency with
defined factors. Nature 451:141–146.
23. Stadtfeld M, Nagaya M, Utikal J, Weir G, Hochedlinger K (2008) Induced pluripotent
stem cells generated without viral integration. Science 322:945–949.
24. Okita K, Nakagawa M, Hyenjong H, Ichisaka T, Yamanaka S (2008) Generation of
mouse induced pluripotent stem cells without viral vectors. Science 322:949 –953.
25. Yu J, et al. (2009) Human Induced Pluripotent Stem Cells Free of Vector and Transgene
Sequences. Science 324:797–801.
26. Zhou H, et al. (2009) Generation of Induced Pluripotent Stem Cells Using Recombinant
Proteins. Cell Stem Cell 4:381–384.
27. Tateishi K, et al. (2008) Generation of insulin-secreting islet-like clusters from human
skin fibroblasts. J Biol Chem 283:31601–31607.
28. Osafune K, et al. (2008) Marked differences in differentiation propensity among
human embryonic stem cell lines. Nat Biotechnol 26:313–315.
29. Chen S, et al. (2009) A small molecule that directs differentiation of human ESCs into
the pancreatic lineage. Nat Chem Biol 5:258 –265.
Maehr et al. PNAS
September 15, 2009
vol. 106
no. 37
Page 6
  • Source
    • "Human induced pluripotent stem cells (iPSCs) technology had been successfully generated via the retrovirus-mediated transfection of four transcription factors (Oct4, Sox2, Klf-4, and c-Myc) (Takahashi et al., 2007; Yu et al., 2007). Several studies had been made to generate iPSCs from patients with various diseases providing new opportunities for regenerative medicine and in vitro disease modeling (Dimos et al., 2008; Park et al., 2008; Maehr et al., 2009). Among disease, those involving retina are necessary and urgent to consider for iPSCs modeling, because of these tissue are not amenable to routine biopsy. "
    [Show abstract] [Hide abstract] ABSTRACT: Age-related macular degeneration (AMD) is one retinal aging process that may lead to irreversible vision loss in the elderly. Its pathogenesis remains unclear, but oxidative stress inducing retinal pigment epithelial (RPE) cells damage is perhaps responsible for the aging sequence of retina and may play an important role in macular degeneration. In this study, we have reprogrammed T cells from patients with dry type AMD into induced pluripotent stem cells (iPSCs) via integration-free episomal vectors and differentiated them into RPE cells that were used as an expandable platform for investigating pathogenesis of the AMD and in-vitro drug screening. These patient-derived RPEs with the AMD-associated background (AMD-RPEs) exhibited reduced antioxidant ability, compared with normal RPE cells. Among several screened candidate drugs, curcumin caused most significant reduction of ROS in AMD-RPEs. Pre-treatment of curcumin protected these AMD-RPEs from H2O2-induced cell death and also increased the cytoprotective effect against the oxidative stress of H2O2 through the reduction of ROS levels. In addition, curcumin with its versatile activities modulated the expression of many oxidative stress-regulating genes such as PDGF, VEGF, IGFBP-2, HO1, SOD2, and GPX1. Our findings indicated that the RPE cells derived from AMD patients have decreased antioxidative defense, making RPE cells more susceptible to oxidative damage and thereby leading to AMD formation. Curcumin represented an ideal drug that can effectively restore the neuronal functions in AMD patient-derived RPE cells, rendering this drug an effective option for macular degeneration therapy and an agent against aging-associated oxidative stress.
    Full-text · Article · Aug 2014 · Frontiers in Aging Neuroscience
  • Source
    • "At present, it is of special interest for the diabetes community to find a way to produce ex vivo pancreatic cell masses to restore biological functions that are lost due to cellular deficits. Because T1D only affects a single cell type, T1D can be treated with novel cellular replacement therapies that are based on reprogramming human embryonic stem cells (hESC) [2] or with human induced pluripotent stem cells (hiPSC) [3], [4] into pancreatic-like cells. However, hESC and hiPSC show several disadvantages, such as ethical problems [5], transgenic strategies [6] or epigenetic failure [7], which limit their use to in vitro assays or preclinical models [8]. "
    [Show abstract] [Hide abstract] ABSTRACT: The conversion of differentiated cells into insulin-producing cells is a promising approach for the autologous replacement of pancreatic cells in patients with type 1 diabetes (T1D). At present, cellular reprogramming strategies encompass ethical problems, epigenetic failure or teratoma formation, which has prompted the development of new approaches. Here, we report a novel technique for the conversion of skin fibroblasts from T1D patients into insulin-expressing clusters using only drug-based induction. Our results demonstrate that skin fibroblasts from diabetic patients have pancreatic differentiation capacities and avoid the necessity of using transgenic strategies, stem cell sources or global demethylation steps. These findings open new possibilities for studying diabetes mechanisms, drug screenings and ultimately autologous transgenic-free regenerative medicine therapies in patients with T1D.
    Full-text · Article · Jun 2014 · PLoS ONE
  • Source
    • "Activin A has been used to induce DE cells in most reports. More recently, it has been shown that the combination of activin A with Wnt3a results in more efficient induction of DE cells11141618212526. In addition to these factors, we chose six growth factors and small molecules from other relevant reports, and examined the differentiation efficiency of DE cells by changing the combinations and concentrations of these factors (Table S1). "
    [Show abstract] [Hide abstract] ABSTRACT: Insulin-producing cells (IPCs) derived from human pluripotent stem cells (hPSCs) may be useful in cell therapy and drug discovery for diabetes. Here, we examined various growth factors and small molecules including those previously reported to develop a robust differentiation method for induction of mature IPCs from hPSCs. We established a protocol that induced PDX1-positive pancreatic progenitor cells at high efficiency, and further induced mature IPCs by treatment with forskolin, dexamethasone, Alk5 inhibitor II and nicotinamide in 3D culture. The cells that differentiated into INSULIN-positive and C-PEPTIDE-positive cells secreted insulin in response to glucose stimulation, indicating a functional IPC phenotype. We also found that this method was applicable to different types of hPSCs.
    Full-text · Article · Mar 2014 · Scientific Reports
Show more