Suppl Tables 1-4 - media.nature.com · 74 SCID-Bg 12 ++ KC 4.5 E+06 38 23 28 na NONE 58 17 17 na 73 21 75 na 119 292 656 2.3 75 SCID-Bg 12 ++ KC

Supplementary Table 1.

Summary of hESC-derived pancreatic endoderm implants

Serum C-peptide (pM)

Mouse No. Strain

Cohort No. GSIS

Graft Site(s)

Total Cell Dose

Time of Assay (days post-implant) Fasting

Glucose Stimulated

Stimulation index Matrix Thymoma

1 SCID-Bg 1 +++ EFP-1

7.4 E+06 63 136 239 1.8 GF+MG

77 279 1026 3.7

91 333 924 2.8

106 319 684 2.1

2 SCID-Bg 1 +++ EFP-1

7.4 E+06 63 105 274 2.6 GF+MG

77 280 1278 4.6

91 185 516 2.8

106 424 943 2.2

118 499 1344 2.7

139 883 3005 3.4

161 397 nd na

210 571 5003 8.8

3 SCID-Bg 1 + EFP-1

7.4 E+06 63 25 124 4.9 GF+MG +

4 SCID-Bg 2 +++ EFP

1.8 E+07 62 226 812 3.6

76 650 1553 2.4

92 577 1719 3.0

110 638 1350 2.1

140 553 865 1.6

5 SCID-Bg 2 +++ EFP

1.8 E+07 62 159 596 3.7 GF+MG

76 480 959 2.0

92 554 1862 3.4

110 931 2196 2.4

140 684 1569 2.3

189 897 4486 5.0

6 SCID-Bg 2 +++ EFP

1.8 E+07 62 149 508 3.4 GF+MG

76 418 1027 2.5

104 323 1091 3.4

7 SCID-Bg 2 +++ EFP

1.8 E+07 62 204 799 3.9 GF+MG

76 513 1527 3.0

92 541 1814 3.4

110 897 2032 2.3

140 592 1726 2.9

189 663 2681 4.0

8 SCID-Bg 3 +++ EFP

1.5 E+07 49 165 576 3.5 GF+MG

66 661 2689 4.1

77 406 1795 4.4

9 SCID-Bg 3 +++ EFP

1.5 E+07 49 116 500 4.3 GF+MG

66 531 2645 5.0

77 404 1569 3.9

10 SCID-Bg 3 +++ EFP

1.5 E+07 49 120 335 2.8 GF+MG

66 544 1737 3.2

77 291 2152 7.4

11 Nude 3 ++ EFP 1.5 E+07 53 93 321 3.4 GF+MG

66 176 431 2.5

83 117 280 2.4

12 Nude 3 ++ EFP 1.5 E+07 53 108 410 3.8 GF+MG

66 207 563 2.7

81 269 698 2.6

13 Nude 3 ++ EFP 1.5 E+07 53 92 289 3.2 GF+MG

66 164 444 2.7

81 251 485 1.9


1.5 E+07 49 225 691 3.1 GF+MG

62 507 1503 3.0

77 597 2651 4.4


1.5 E+07 45 131 629 4.8 GF+MG

62 578 4211 7.3

73 701 2613 3.7


1.5 E+07 49 159 458 2.9 GF+MG

62 969 3816 3.9

79 501 1573 3.1

17 SCID-Bg 4 ++ EFP

1.5 E+07 49 162 400 2.5 GF+MG +

62 225 681 3.0

77 129 377 2.9


1.5 E+07 49 206 360 1.7 GF+MG

62 408 1270 3.1

77 704 2640 3.8

92 519 1843 3.6


1.5 E+07 49 228 826 3.6 GF+MG

62 616 1406 2.3

77 777 2713 3.5


1.5 E+07 49 111 316 2.8 GF+MG

62 207 591 2.9

77 644 2525 3.9


1.5 E+07 49 97 258 2.7 GF+MG +

62 169 704 4.2

77 92 267 2.9

22 Nude 4 - EFP 1.5 E+07 49 22 32 na GF+MG

62 12 12 na

77 6 17 na

23 Nude 4 - EFP-1 7.4 E+06 49 24 34 na GF+MG

62 37 11 na

77 4 22 na


1.5 E+07 42 81 233 2.9 GF+MG

54 170 708 4.2

69 176 433 2.5


1.5 E+07 42 123 658 5.4 GF+MG

54 233 1418 6.1

69 371 806 2.2


1.5 E+07 42 182 507 2.8 GF+MG

54 299 2171 7.3

69 332 840 2.5


1.5 E+07 42 142 895 6.3 GF+MG

54 296 3912 13.2

69 471 1205 2.6

28 SCID-Bg 5 ++ EFP

1.5 E+07 42 34 43 na GF+MG

54 69 263 3.8

69 105 315 3.0

82 91 563 6.2


1.5 E+07 42 136 643 4.7 GF+MG

54 288 1540 5.3

69 253 707 2.8


1.5 E+07 42 133 575 4.3 GF+MG

54 215 1040 4.8

69 301 1096 3.6


1.5 E+07 42 153 505 3.3 GF+MG

54 302 2278 7.5

69 275 1304 4.7


1.5 E+07 42 122 347 2.8 GF+MG

54 414 1156 2.8

69 257 1050 4.1


1.5 E+07 42 163 393 2.4 GF+MG

54 351 1268 3.6

69 429 1455 3.4


1.5 E+07 37 72 221 3.0 GF+MG

49 157 670 4.3

64 206 991 4.8

106 672 2300 3.4


1.5 E+07 37 57 242 4.2 GF+MG

49 169 491 2.9

64 239 676 2.8


1.5 E+07 30 7 19 na GF+MG

44 94 202 2.2

71 482 1153 2.4

94 611 2625 4.3

142 870 5133 5.9


1.5 E+07 30 18 35 na GF+MG

44 126 825 6.6

71 698 1862 2.7

94 679 3067 4.5

142 1557 3645 2.3


1.5 E+07 30 19 12 na GF+MG +

46 126 297 2.4

71 373 868 2.3

94 498 1599 3.2


1.5 E+07 30 26 43 na GF+MG

44 152 463 3.0

71 333 2930 8.8

94 590 1632 2.8

144 678 4406 6.5


1.5 E+07 30 35 75 na GF+MG

44 113 1696 15.0

71 512 2366 4.6

94 656 2775 4.2

144 1716 5160 3.0


1.5 E+07 30 37 87 na GF+MG

44 159 1248 7.9

71 578 2588 4.5

94 701 1773 2.5


1.5 E+07 30 33 103 3.1 GF+MG

44 215 1327 6.2

71 566 2753 4.9

94 468 1556 3.3

43 SCID-Bg 7 - EFP

1.5 E+07 30 11 21 na GF+MG +

44 40 54 na


1.5 E+07 30 18 51 na GF+MG

44 226 548 2.4

71 835 2896 3.5

94 722 3856 5.3


1.5 E+07 30 23 68 na GF+MG

44 159 352 2.2

71 1082 2961 2.7

94 964 2520 2.6


1.5 E+07 50 18 37 na GF+MG

84 156 495 3.2

114 490 1575 3.2


1.5 E+07 50 20 43 na GF+MG

84 195 540 2.8

114 980 3151 3.2


1.2 E+07 50 6 45 na GF+MG

84 102 332 3.3

114 681 2203 3.2


1.5 E+07 50 23 54 na GF+MG

84 322 746 2.3

114 966 2461 2.5


1.5 E+07 50 15 29 na GF+MG

84 125 315 2.5

114 1145 2417 2.1


1.5 E+07 60 501 1569 3.1 GF+MG


1.5 E+07 60 589 1647 2.8 GF+MG

53 SCID-Bg 10a +++ EFP

1.5 E+07 56 512 1003 2.0 GF+MG


1.5 E+07 56 280 933 3.3 GF+MG


1.5 E+07 60 278 517 1.9 GF+MG

83 426 815 1.9


1.5 E+07 60 409 805 2.0 GF+MG

57 SCID-Bg 10a ++ EFP

1.5 E+07 60 101 217 2.1 GF+MG

58 SCID-Bg 10b - SC

1.5 E+07 59 38 41 na GF+MG +

59 SCID-Bg 10b +++ SC

1.5 E+07 59 208 553 2.7 GF+MG

80 472 850 1.8

60 SCID-Bg 10b ++ SC

1.5 E+07 57 150 351 2.3 GF+MG +


1.5 E+07 59 36 59 na

80 126 340 2.7

62 SCID-Bg 10b + SC

1.5 E+07 59 17 32 na GF+MG

80 46 140 3.1


1.5 E+07 59 60 228 3.8 GF+MG

80 99 305 3.1


1.5 E+07 34 15 38 na MG

75 324 2184 6.7


1.5 E+07 34 32 36 na MG

75 572 3777 6.6

66 SCID-Bg 11 +++ EFP-1

7.4 E+06 34 17 40 na MG

75 456 2172 4.8

67 SCID-Bg 11 +++ EFP-1

7.4 E+06 34 20 33 na NONE

75 508 1814 3.6


1.5 E+07 34 31 69 na GF

75 432 3436 8.0


1.5 E+07 34 26 105 4.1 GF

75 590 2438 4.1


1.5 E+07 34 30 109 3.6 GF +

75 496 1577 3.2


1.5 E+07 34 25 68 na GF+MG

75 527 2335 4.4


1.5 E+07 34 25 59 na GF+MG

75 799 2391 3.0

73 SCID-Bg 11 + EFP

1.5 E+07 34 23 57 na GF+MG +

75 62 132 2.1

74 SCID-Bg 12 ++ KC

4.5 E+06 38 23 28 na NONE

58 17 17 na

73 21 75 na

119 292 656 2.3

75 SCID-Bg 12 ++ KC

4.5 E+06 38 24 27 na NONE

58 35 58 na

73 88 233 2.7

76 SCID-Bg 12 ++ KC

7.4 E+06 38 26 39 na NONE

58 47 80 na

73 94 261 2.8

77 SCID-Bg 12 ++ KC

7.4 E+06 38 25 36 na NONE

58 72 157 2.2

73 156 451 2.9

78 SCID-Bg 12 +++ KC

7.4 E+06 38 27 40 na NONE

58 92 166 1.8

73 209 721 3.4

79 SCID-Bg 12 +++ KC

6.7 E+06 38 30 82 na NONE

58 144 565 3.9

73 247 920 3.7

80 SCID-Bg 13 + EFP-1

7.4 E+06 37 51 59 na GF+MG

57 80 82 na

72 61 139 2.3

81 SCID-Bg 13 +++ EFP-1

7.4 E+06 37 32 72 na GF+MG

57 130 1012 7.8

72 244 730 3.0

82 SCID-Bg 13 ++ EFP-1

5.4 E+06 37 28 48 na GF+MG

57 82 425 5.2

72 174 584 3.4

83 SCID-Bg 13 +++ EFP-1

5.4 E+06 37 37 51 na GF+MG

57 117 409 3.5

72 297 836 2.8

84 SCID-Bg 13 +++ EFP-1

5.4 E+06 37 42 68 na GF+MG

57 175 498 2.8

72 357 1597 4.5

85 SCID-Bg 13 +++ EFP-1

5.4 E+06 37 36 81 na GF+MG

57 159 641 4.0

72 328 1352 4.1


1.3 E+07 37 55 215 3.9 GF

57 175 1674 9.6

72 375 1566 4.2


1.3 E+07 37 54 134 2.5 GF

57 242 536 2.2

72 318 1922 6.1

88 Nude 14a ++ EFP 1.5 E+07 60 241 375 1.6 GF+MG

70 253 477 1.9

89 Nude 14a ++ EFP 1.5 E+07 60 144 318 2.2 GF+MG

70 153 299 2.0

90 Nude 14a ++ EFP 1.5 E+07 60 88 150 1.7 GF+MG

70 218 374 1.7

91 Nude 14a ++ EFP 1.5 E+07 60 195 301 1.5 GF+MG

70 282 363 1.3

92 Nude 14a ++ EFP 1.5 E+07 60 348 447 1.3 GF+MG

70 279 441 1.6

93 Nude 14b - SC 1.5 E+07 62 13 15 na GF+MG

94 Nude 14b - SC 1.5 E+07 62 21 50 na GF+MG

95 Nude 14b - SC 1.5 E+07 62 20 33 na GF+MG

96 Nude 14b - SC 1.5 E+07 62 20 71 na GF+MG

97 Nude 14b - SC 1.5 E+07 60 11 28 na GF+MG

98 SCID-Bg 14c ++ SC

1.5 E+07 59 23 53 na GF+MG

80 37 111 3.0

113 128 386 3.0


1.5 E+07 59 23 49 na GF+MG

80 42 18 na

113 229 481 2.1


1.5 E+07 59 17 35 na GF+MG

80 26 87 na

113 49 344 6.9


1.5 E+07 59 30 104 3.5 GF+MG

80 74 465 6.3


1.5 E+07 59 20 35 na GF+MG

80 31 54 na

113 93 492 5.3

103 SCID-Bg 14c +++ SC

1.5 E+07 59 79 244 3.1 GF+MG

80 229 814 3.6

104 SCID-Bg 15 ++ EFP

1.5 E+07 53 243 452 1.9 GF+MG

77 574 2237 3.9

105 SCID-Bg 15 ++ EFP

1.5 E+07 53 253 619 2.4 GF+MG

77 437 2620 6.0

Key

GSIS + C-peptide 100-200 pM

++ C-peptide 200-700 pM

+++ C-peptide >700 pM

- C-peptide <100 pM

Implant Sites EFP Epididymal Fat Pad, two implants per mouse

EFP-1 Epididymal Fat Pad, one implant

KC Kidney Capsule

SC Abdominal area subcutaneous

Matrix GF Gelfoam

MG Matrigel

na not applicable

nd not determined

Thymoma + Mouse had enlarged thymus at time of sacrifice

This is frequently associated with immune reactivation

Statistics

All Mice SCID EFP Implants

GSIS No. of mice %

No. of mice %

- 9 8.6 1 1.4

+ 4 3.8 3 4.2

++ 27 25.7 6 8.3

+++ 65 61.9 62 86.1

ALL + 96 91.4 71 98.6

Total 105 72

In addition to the epididymal fat pad (EFP), implant sites tested included the kidney capsule and abdominal subcutaneous space. Kidney capsule (KC) engraftment efficiency was similar to EFP engraftment with respect to the generation of glucose responsive cells, but high levels of serum C-peptide (both fasting and glucose-stimulated) appeared slightly earlier in EFP engrafted mice. For example, after 70 days of engraftment the glucose-stimulated serum C-peptide levels in EFP engrafted mice (No. 82-85) ranged 580-1600 pM, while mice (No. 76-79) that received a similar dose of cells in the kidney capsule ranged from 260-920 pM.

The subcutaneous (SC) site was also supportive of engraftment and development of functional cells, but less efficient (see mice from Cohort No. 10). By 56-60 days post-implant, four of the five EFP-implanted mice had glucose-stimulated serum C-peptide levels ranging from 500-1000 pM, while by 80 days post-implant only one of four SC-implanted mice (No. 59) reached these levels. Eventually, highly functional grafts were obtained in the majority of the subcutaneously implanted SCID mice (also see Cohort 14c). After prolonged implantation, the efficiency of functional engraftment in SCID-Bg mice (as defined by glucose-stimulated serum C-peptide levels over 200 pM, was 83% (10 of 12 mice) for subcutaneous implants, 100% (6 of 6 mice) for kidney capsule implants, and 94% (68 of 72 mice) for EFP implants. These numbers include the animals that present with thymoma or lymphoma, which typically occurs in 10-30% of SCID-Bg mice and is linked to immune reactivation

1, 2.

The total cell dose that was implanted in the various experiments ranged from 4.5x106 to

1.8x107. It appears that at the lower implant doses, the levels of serum C-peptide

detected at the earliest measurements are slightly lower. For example compare C-peptide levels at about 60 days post implant for mouse No. 2 (one graft, total 7.4 x10

6

cells; basal and glucose-stimulated serum C-peptide levels of 105 and 274 pM, respectively) and mouse No. 5 (two grafts, total 1.8 x10

7 cells; basal and glucose-

stimulated serum C-peptide levels of 159 and 596 pM, respectively). At later measurements the levels of C-peptide in the mice implanted with lower doses of cells appear similar to the mice implanted with higher doses.

The methods of cell delivery were identical for the SC and EFP implant sites, but the cell aggregates were implanted without Gelfoam or Matrigel in the KC site, suggesting that neither was required for successful engraftment. Confirming this, functional hESC-derived grafts were obtained with cells implanted in the EFP site without Gelfoam (mice Nos. 64-66), without Matrigel (mice Nos. 68-70, 86, 87), or without either Gelfoam or Matrigel (mouse No. 67).

Functional engraftment was assessed in two different immune-compromised mouse strains, SCID-beige (C.B-17/IcrHsd-Prkdc

scid Lyst

bg) and Athymic Nude (Nude-FOXN1

NU).

In a cohort of 10 implanted mice (Cohort 4), the two Nude mice (Nos. 22, 23) never engrafted, while highly functional grafts developed in all eight SCID-Bg mice (mice Nos. 14-21). In addition, no successful engraftment was observed in five Nude mice (Cohort 14b) implanted subcutaneously. In another cohort of mice (Cohort 3) functional EFP grafts were obtained in Nude mice (mice Nos. 11-13), but total C-peptide levels were lower compared to SCID-Bg mice (Nos. 8-10) implanted at the same time with the same source and dose of cells.

References 1. Bosma, G.C. et al. Evidence of functional lymphocytes in some (leaky) scid mice.

J Exp Med 167, 1016-1033 (1988). 2. Inohara, H. et al. Reduction of leaky lymphocyte clones producing

immunoglobulins and thymic lymphocytic leukemia by selective inbreeding of SCID (severe combined immunodeficiency) mice. Acta Otolaryngol Suppl 501, 107-110 (1993).

Supplementary Table 2. Molar ratios of proinsulin, insulin and C-peptide in engrafted animals. Mouse No.

(Serum) Proinsulin (pM)

Insulin (pM)

Ratio insulin to proinsulin

% processed

C-peptide (pM)

Ratio C-peptide to proinsulin

% processed

8 25 697 28 96.5 2654 105 99.1

9 14 448 33 97.1 2210 163 99.4

10 10 380 39 97.5 1624 167 99.4

11 10 329 33 97.0 632 62 98.4

12 7 160 23 95.8 916 131 99.2

14 28 1300 47 97.9 3472 125 99.2

15 17 686 41 97.6 1872 111 99.1

16 17 1037 60 98.3 2383 137 99.3

18 19 371 19 95.1 2437 128 99.2

19 16 772 48 98.0 2801 174 99.4

20 13 751 59 98.3 2149 169 99.4

24 9 246 28 96.5 937 106 99.1

25 15 963 64 98.5 1843 122 99.2

26 9 457 50 98.0 1328 144 99.3

27 18 1201 66 98.5 1825 100 99.0

29 11 510 45 97.8 2481 218 99.5

30 13 343 26 96.3 1835 139 99.3

31 1 205 236 99.6 1362 1573 99.9

32 13 508 40 97.5 1670 130 99.2

33 13 589 47 97.9 2038 161 99.4

35 3 255 75 98.7 919 269 99.6

Average 97.5 99.3

Standard Deviation 1.1 0.3

(Graft portion)

35 6.6 pmol 224 pmol 34 97.1 168 pmol 25 96.2

Supplementary Table 3 Histopathology analysis of hES-cell derived pancreatic endoderm grafts.

1 - Tissue with an admixture of cell types derived from multiple germ layers exhibiting the characteristics of a teratoma.

Incidence % of Grafts

Ectoderm Mesoderm Endoderm

>70% Endocrine

36/46 78.3%

<30% Duct Cells

35/46 76%

Teratomatous Elements 1

6/46 13% No Yes Yes

Teratoma 1/46 2.2% Yes Yes Yes

Supplementary Table 4 QPCR primer sequences for human genes analyzed in Supplementary Figure 1.

Gene Forward primer Reverse primer

CYCG 5'-CTTGTCAATGGCCAACAGAGG 5'-GCCCATCTAAATGAGGAGTTGGT

TBP 5'-TGTGCACAGGAGCCAAGAGT 5'-ATTTTCTTGCTGCCAGTCTGG

BRACH 5'-TGCTTCCCTGAGACCCAGTT 5'-GATCACTTCTTTCCTTTGCATCAAG

CER 5'-ACAGTGCCCTTCAGCCAGACT 5'-ACAACTACTTTTTCACAGCCTTCGT

FOXA2 5'-GGGAGCGGTGAAGATGGA 5'-TCATGTTGCTCACGGAGGAGTA

HNF1B 5'-TCACAGATACCAGCAGCATCAGT 5'-GGGCATCACCAGGCTTGTA

HNF4A 5'-CATGGCCAAGATTGACAACCT 5'-TTCCCATATGTTCCTGCATCAG

SOX9 5'-AGTACCCGCACTTGCACAAC 5'-ACTTGTAATCCGGGTGGTCCTT

HNF6 5'-CGCTCCGCTTAGCAGCAT 5'-GTGTTGCCTCTATCCTTCCCAT

PDX1 5'-AAGTCTACCAAAGCTCACGCG 5'-GTAGGCGCCGCCTGC

PROX1

5'-AACATGCACTACAATAAAGCAAATGAC 5'-CAGGAATCTCTCTGGAACCTCAAA

PTF1A 5'-GAAGGTCATCATCTGCCATCG 5'-GGCCATAATCAGGGTCGCT

NKX6-1 5'-CTGGCCTGTACCCCTCATCA 5'-CTTCCCGTCTTTGTCCAACAA

NGN3 5'-GCTCATCGCTCTCTATTCTTTTGC 5'-GGTTGAGGCGTCATCCTTTCT

NKX2-2 5'-GGCCTTCAGTACTCCCTGCA 5'-GGGACTTGGAGCTTGAGTCCT

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Suppl Tables 1-4 - media.nature.com · 74 SCID-Bg 12 ++ KC 4.5 E+06 38 23 28 na NONE 58 17 17 na 73 21 75 na 119 292 656 2.3 75 SCID-Bg 12 ++ KC