1. Journal Club 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 2009 年９月 11 日 8:30-8:55 ８階 医局 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 double- blind 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

2. GLP-1

5. a nasal spray formulation of recombinant human GLP-1 a stabilized GLP-1 analogue

6. 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.

7. Diabetes Care 32:1237–1243, 2009 the 1 Diabeteszentrum, Bad Lauterberg im Harz, Germany; the 2 Medstar Research Institute, Hyattsville, Maryland; the 3 Profil Institute, Neuss, Germany; the 4 Roche Laboratories, Nutley, New Jersey; and 5 Hoffmann-La Roche, Basel, Switzerland.

8. 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.

9. 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.

11. Table 1—Baseline demographics, disease characteristics, and changes after the 8-week treatment

12. 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.

13. 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.

14. 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.

15. 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.

17. 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, mild- to-moderate nausea; the incidence of hypoglycemia was very low.

18. CONCLUSIONS Taspoglutide used in combination with metformin significantly improves fasting and postprandial glucose control and induces weight loss, with a favorable tolerability profile.

19. 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

22. 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３ １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」

23. www.pnas.orgcgidoi10.1073pnas.0906894106

24. 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). 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.

25. 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.

26. 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, 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, whereas DiPS lines H2.1, H1.1, H1.5 were generated without addition of the chemical. Colonies were picked starting ~4 weeks after infection.

27. 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.

28. 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×L- Glu, 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.

29. METHODS 5 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 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.

30. 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 Whole- GenomeExpression 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.

31. 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 C- peptide ELISA kit (Alpco Diagnostics).

33. 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.

34. 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.

35. 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.

36. Fig. 2. Expression analysis of patient specific DiPS cells. (B) Hierarchical cluster analysis of different DiPS, HUES and fibroblast lines.

37. 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.

38. 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.

39. 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).

40. Fig. 4. Stepwise differentiation of ES/DiPS cells toward beta-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.

41. 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.

43. 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, somatostatin.

45. 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.

46. 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？！