Transcript B - 埼玉医科大学総合医療センター 内分泌・糖尿病内科

Journal Club
Nauck MA, Ratner RE, Kapitza C, Berria R, Boldrin M, Balena R.
Treatment with the human once-weekly glucagon-like peptide-1 analog taspoglutide in
combination with metformin improves glycemic control and lowers body weight in
patients with type 2 diabetes inadequately controlled with metformin alone: a doubleblind placebo-controlled study.
Diabetes Care. 2009 Jul;32(7):1237-43. Epub 2009 Apr 14.
René Maehr, Shuibing Chen, Melinda Snitow, Thomas Ludwig, Lisa Yagasaki, Robin
Goland, Rudolph L. Leibel, and Douglas A. Melton
Generation of pluripotent stem cells from patients with type 1 diabetes
PNAS published online before print August 31, 2009, doi:10.1073/pnas.0906894106
2009年９月11日 8:30-8:55
８階 医局
埼玉医科大学 総合医療センター 内分泌・糖尿病内科
Department of Endocrinology and Diabetes,
Saitama Medical Center, Saitama Medical University
松田 昌文
Matsuda, Masafumi
GLP-1
a nasal spray formulation of
recombinant human GLP-1
a stabilized GLP-1 analogue
Taspoglutide (R1583/BIM51077) is a human GLP-1 analog with a
pharmacokinetic profile suitable for weekly subcutaneous
administration, through two amino acid substitutions in positions 8 and
35 with aminoisobutyric acid and a sustained release formulation.
The compound has been licensed exclusively to Roche for development and marketing worldwide except Japan,
where it will be comarketed with Teijin, and France, where Ipsen retains the right to comarket the product.
the 1Diabeteszentrum, Bad Lauterberg im Harz, Germany; the 2Medstar
Research Institute, Hyattsville, Maryland; the 3Profil Institute, Neuss,
Germany; the 4Roche Laboratories, Nutley, New Jersey; and 5Hoffmann-La
Roche, Basel, Switzerland.
Diabetes Care 32:1237–1243, 2009
BACKGROUND
To evaluate the efficacy and
safety of taspoglutide
(R1583/BIM51077), a human
once-weekly glucagon-like
peptide-1 analog, in patients
with type 2 diabetes
inadequately controlled with
metformin.
METHODS
Type 2 diabetic (n = 306) patients who failed to
obtain glycemic control (A1C 7–9.5%) despite
1,500 mg metformin daily were randomly
assigned to 8 weeks of double-blind
subcutaneous treatment with placebo or
taspoglutide, either 5, 10, or 20 mg once
weekly or 10 or 20 mg once every 2 weeks,
and followed for 4 additional weeks. All
patients received their previously established
dose of metformin throughout the study.
Glycemic control was assessed by change in
A1C (percent) from baseline.
Table 1—Baseline demographics, disease characteristics, and changes after the 8-week treatment
Figure 2—Effects of taspoglutide and placebo on A1C. All taspoglutide doses
were statistically significant (P<0.0001) (A). Black, placebo; magenta, 5 mg once
weekly; green, 10 mg once weekly; yellow, 20 mg once weekly; purple, 10 mg once
every 2 weeks; orange, 20 mg once every 2 weeks.
Figure 2—Effects of taspoglutide and placebo on Percentage of patients achieving
target A1C, *P < 0.0001 vs. placebo.
Black, placebo; magenta, 5 mg once weekly; green, 10 mg once weekly; yellow, 20
mg once weekly; purple, 10 mg once every 2 weeks; orange, 20 mg once every 2
weeks.
Figure 2—Effects of taspoglutide and placebo on fasting plasma glucose (C);
Black, placebo; magenta, 5 mg once weekly; green, 10 mg once weekly; yellow, 20
mg once weekly; purple, 10 mg once every 2 weeks; orange, 20 mg once every 2
weeks.
Figure 2—Effects of taspoglutide and placebo on body weight (D). Black, placebo;
magenta, 5 mg once weekly; green, 10 mg once weekly; yellow, 20 mg once
weekly; purple, 10 mg once every 2 weeks; orange, 20 mg once every 2 weeks.
RESULTS
Significantly greater (P<0.0001) reductions in A1C from a
mean ± SD baseline of 7.9 ± 0.7% were observed in all
taspoglutide groups compared with placebo after 8 weeks of
treatment: –1.0 ± 0.1% (5 mg once weekly), –1.2 ± 0.1% (10
mg once weekly), –1.2 ± 0.1% (20 mg once weekly), –0.9 ±
0.1% (10 mg Q2W), and –1.0 ± 0.1% (20 mg Q2W) vs. –0.2 ±
0.1% with placebo. After 8 weeks, body weight loss was
significantly greater in the 10 mg (–2.1 ± 0.3 kg, P<0.0035 vs.
placebo) and 20 mg (–2.8 ± 0.3 kg, P_0.0001) once-weekly
groups and the 20 mg once every 2 weeks (–1.9 ± 0.3 kg,
P<0.0083) group than with placebo (– 0.8 ± 0.3 kg). The most
common adverse event was dose-dependent, transient, mildto-moderate nausea; the incidence of hypoglycemia was very
low.
CONCLUSIONS
Taspoglutide used in
combination with metformin
significantly improves fasting
and postprandial glucose control
and induces weight loss, with a
favorable tolerability profile.
Message
Taspoglutideでは２週間に一度の注射でOK？！
だけじゃない
テイジン
米ハーバード大ダグラス・メルトン教授らのグループが、インスリンを分泌する膵臓（すいぞう）
の「ベータ細胞」が破壊されてしまう１型糖尿病の解明と治療に向け、患者自身の皮膚細胞か
ら万能細胞「ｉＰＳ細胞」をつくり出すことに成功したと、８月３１日付の米国科学アカデミー紀要
（電子版）で発表した。
グループはまた、このｉＰＳ細胞を、インスリンを生産する機能を持つ細胞に変えることにも成
功したとしている。専門家からは、患者自身の細胞から拒絶反応のないベータ細胞をつくり、
体内に戻す再生医療に道筋を付けるものと評価の声が出ている。
同教授らは病気の再現も試みており、矢ケ崎さんは信濃毎日新聞の取材に「１型糖尿病の
発生解明に向けた第一歩」としている。
グループは、１型糖尿病を発症した３０代の白人男性２人から皮膚細胞を採取。そこに３種
類の遺伝子を組み込むことでｉＰＳ細胞に変えた。さらにベータ細胞と同様の機能を持つ細胞
へと育て、試験管でグルコース（糖）を与えたところ、グルコース濃度が高まると盛んにインスリ
ンが分泌され、正常に働いていることが確認されたという。
「ｉＰＳ細胞から数種類の細胞をつくってマウスに移植すると、人為的に１型糖尿病を発症させ
ることができる。糖尿病発生のメカニズムを観察すれば、根本的な原因を解明できるのでは」
「拒絶反応がなく機能するインスリン産生細胞を、『オーダーメード』で供給する方法が確立さ
れたと言える。実際の応用には課題も残るが、再生医療による糖尿病治療のゴールが見えて
きたと言っても過言ではない」
www.pnas.orgcgidoi10.1073pnas.0906894106
BACKGROUND AND OVERVIEW
Type 1 diabetes (T1D) is the result of an autoimmune
destruction of pancreatic β 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). T1Dspecific 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.
METHODS 1
Cell Culture. Skin fibroblasts from T1D patients and controls were derived
from explants of 3-mm dermal biopsies. Briefly,3-mmskin 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% CO2). 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
medium (DMEM supplemented with 10% FBS, glutamine, sodium pyruvate,
nonessential amino acids, and penicillin/streptomycin). The resulting
fibroblasts 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, _mercaptoethanol, 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.
METHODS
2
Reprogramming. VSVG-coated retroviruses were generated
according to standard 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). The media was
changed on day 3 to DMEM supplemented with 10% FBS, Lglutamine, 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, whereas
DiPS lines H2.1, H1.1, H1.5 were generated without addition
of the chemical. Colonies were picked starting ~4 weeks after
infection.
METHODS 3
Spontaneous Differentiation. Spontaneous differentiation
through EB formation was initiated by dissociation of human
DiPS cells using collagenase IV treatment, and subsequent
transfer to low attachment 6-well plates in knockout DMEM
supplemented with 20% knockout serum replacement,
nonessential amino acid, beta-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.
METHODS 4
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 ActivinA(R&D systems)
in advanced RPMI (A-RPMI; Invitrogen) supplemented with 1×L-Glu and
1_PS for 1 day, followed by treatment with 100 ng/mL Activin A in A-RPMI
supplemented with 1×L-Glu, 1×PS and 0.2% FBS (Invitrogen). Two days
later, the media was changed to 50 ng/mL FGF10 (R&D systems) ＋0.25
μM KAAD-CYC (Calbiochem) in A-RPMI supplemented with 1×L-Glu,
1×PS and2%FBS and maintained for additional 2 days. Cells were then
transferred to 50 ng/mL FGF10 ＋ 0.25μMKAAD-CYC ＋ 2 μMRA (Sigma) in
DMEM supplemented with 1×L-Glu, 1×PS, 1× B27 (Invitrogen) 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 1×LGlu, 1×PS, 1× B27 and cultured for an additional 4 days. Then, cells
were transferred to 50 ng/mL EX-4 (Sigma) ＋ 10 μM DAPT (Sigma) in
DMEM supplemented with 1×L-Glu, 1×PS, 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 1×L-Glu, 1×PS, 1× B27 for 6 days.
METHODS 5
Immunofluorescence. Immunofluorescence staining
was performed using primary antibodies against Cpeptide (4020-01; Linco), FOXA2 (07-633; Upstate),
glucacon (4031; Linco), HNF6 (sc-13050; Santa Cruz
Biotechnology), 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 Molecular Probes.
METHODS 6
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 WholeGenomeExpression BeadChips (HumanRef-8)wasfollowed by
analysison an Illumina Beadstation 500. All samples were
prepared in duplicates. Data analysis was conducted using
manufacturer’s Beadstudio software.
METHODS 7
C-Peptide Release Assay. C-peptide release was measured by
incubating the cells in Krebs–Ringer solution containing
bicarbonate and Hepes (KRBH; 129 mM NaCl/4.8 mM KCl/2.5
mM CaCl2/1.2 mM KH2PO4/1.2 mM MgSO4/5 mM
NaHCO3/10mMHepes/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 Cpeptide ELISA kit (Alpco Diagnostics).
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.
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.
Fig. 2.
Expression
analysis of
patient specific
DiPS cells.
(A)
Semiquantitative
analysis of
expression of
OCT4, SOX2,
REX1,NANOG,K
LF4, GDF3, and_
ACTIN.
Control PCR (no
RT) is included.
Fig. 2. Expression analysis of patient specific DiPS cells.
(B) Hierarchical cluster analysis of different DiPS, HUES and
fibroblast lines.
Fig. 2. Expression analysis of
patient specific DiPS cells.
(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.
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.
Fig. 3. Spontaneous differentiation of DiPS cells into cells of different germ layer
origin
(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 beta-like cells. (A)
Schematic representation of stepwise differentiation of human PS cells to betalike 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.
Fig. 4. Stepwise
differentiation of ES/DiPS
cells toward _-like cells.
(A) Schematic
representation of stepwise
differentiation of human PS
cells to beta-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.
Fig. 4. Stepwise
differentiation of ES/DiPS
cells toward _-like cells.
(A) Schematic
representation of
stepwise differentiation of
human PS cells to betalike 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.
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 Dglucose, and the amount of human C-peptide released to culture supernatant was
analyzed by ELISA. C-PEP, C-peptide; INS, insulin; GLU, glucagon; SS,
somatostatin.
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 DiPSderived 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, somatostatin.
SUMMARY
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.
Message
１型糖尿病のモデル細胞ができた。
その細胞がインスリン産生する細胞になる。
将来の１型糖尿病患者の治療に使える？！