Table S1. Real-time PCR primer sequences.

Target Forward primer Reverse primer base pair

References

CD24 AACTAATGCCACCACCAAGG GGACTTCCAGACGCCATTTG 167 NM_013230.3

GATA3 AGGCAGGGAGTGTGTGAACT CTTCGCTTGGGCTTAATGAG 141 NM_001002295.1

PROM1 (CD133)

ACCGACTGAGACCCAACATC GACCGCAGGCTAGTTTTCAC 172 NM_001145847.1

THBS2 ACCTCCTGTCCCTTGACCTT ATGCCCAATTTTCACTCCAC 173 NM_003247.3

LY96 CTGAAGGGAGAGACTGTGAA GGTTGGTGTAGGATGACAAA 149 NM_015364.4

IL1R1 GGGACTTTACACAGGGACCA TCCTTAGTGGCTGGTGACAG 124 NM_000877.3

CXCR4 GGAAAAGATGGGGAGGAGAG AAGAGGCAAAGGAATGGACA 181 NM_001008540.1

IFIT3 GACGGGTAGGACGATAGGAA TGTTAGCCAGGATGGTCTCA 199 NM_001549.5

FGFR3 ACCTGAAGATGGGAGCCTTT TCCGTTGTACCAGCCTTTTC 145 NM_000142.4

IDO1 TATGACGCCTGTGTGAAAGC TCAGTGCCTCCAGTTCCTTT 155 NM_002164.5

KDR AAGTCGGGAGAGGAGAGAGG CACATGGATTGGCAGAGGCT 182 NM_002253

GAPDH ACAGTCAGCCGCATCTTCTT GGCAACAATATCCACTTTACC 151 NM_002046.5

Figure S1. The expression profiles of somatic cells, hESCs (NTU1) and hiPSCs (iCFB). Genes involved in human immune system and demonstrated significant differential expressions between somatic and pluripotent cells are enlisted. PGCs: primary granulosa cells, PDPCs: primary dermal papilla cells, PFCs: primary foreskin fibroblast cells, iCFBD0: undifferentiated iCFB hiPSCs, NTU1D0: undifferentiated NTU1 hESCs, iCFBD15: 15 days differentiated iCFB hiPSCs and NTU1D15: 15 days differentiated NTU1 hESCs.

(A)

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(D)

Figure S2. The top 20 up- or down-regulated genes were identified in pluripotent stem cells. Undifferentiated stem cells of (A) NTU1 and (B) iCFB, and differentiated stem cells of (C) NTU1 and (D) iCFB were compared with PDPCs, PFCs and PGCs.

iC F B v s P D P CF

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Figure S3. The genetic expression profiles of somatic cells, hESCs (NTU1) and hiPSCs (iCFB) were shown in human immune system (Immunome database and our lab selected genes were enlisted). PGCs: primary granulosa cells, PDPCs: primary dermal papilla cells, PFCs: primary foreskin fibroblast cells, iCFBD0: undifferentiated iCFB hiPSCs, NTU1D0: undifferentiated NTU1 hESCs, iCFBD15: 15 days differentiated iCFB hiPSCs and NTU1D15: 15 days differentiated NTU1 hESCs.

(A)

(B)

Figure S4. The surface expression of CD24 was decreased by rapamycin. (A) H9 differentiated stem cells were treated with 50 ng/ml Rapamycin and analyzed by flow cytometry and (B) CD24 mean value was significantly reduced by rapamycin treatment. Mean ± SEM is based on 6 replicate experiments. using different batches of differential stem cells. *P<0.05.

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CD4+ CD25

+

5.35%

Control Rapamycin

CD4+ CD25

+

6.61%

(D)

Figure S5. The action of rapamycin affects CD3+CD4+CD25+ human regulatory T cells (Treg) activation and function. (A) The percentage of Treg cells and (B) the expression levels of IL-10 (C) TGFβ and (D) neutralized antibodies against to IL-10 and TGFβ in 15-day rapamycin-treated differentiated stem cells co-cultured with PBMCs compared with the control differentiated stem cells. Mean ± SEM is based on 3–5 replicate experiments using H9 differential stem cells. *P<0.05 and **P<0.01.

Table S2A. The up-regulated genes in undifferentiated stem cells compared to somatic cells.

Comparison (Fold Change) Ranking (Top 10)

Compare to PDPC

Compare to PFC

Compare to PGC

Compare to PDPC

Compare to PFC Compare to PGC

GeneID NTU iCFB NTU iCFB NTU iCFB NTU iCFB NTU iCFB NTU iCFB

ARG2 3.284 3.383 5.307 5.406 3.447 3.545

5 6

BMP7 4.166 4.633 3.636 4.103 3.954 4.421

8

6 6

CALCB

4.287

4.565

3.823

CD24 5.773 5.819 9.653 9.699 5.512 5.559 2 3 1 1 1 1

CLU 4.409 4.726 -1.286 -0.922 0.976 1.327 10 7

CR1L

4.017

4.090

4.060

7

CXCL12

-0.495

-3.747

3.681

CXCL5 5.682 6.587 3.278 4.182 1.764 2.668 3 2

CXCL6 3.994

-1.669

-4.305

EMB 1.800 1.972 4.452 4.624 1.897 2.069

10

FGFR1 2.502 2.872 2.584 3.242 3.773 3.834

9 10

FGFR2 3.079 3.345 5.236 5.501 3.141 3.406

6 5

FGFR3 5.057 4.566 6.017 5.526 3.735 3.244 4 9 3 4 10

FGFR4

4.437

4.165

3.502

HMMR 3.989 4.432 4.376 4.819 1.531 1.974

9

IDO1 4.502 5.030 4.453 4.980 4.513 5.040 9 6

8 4 3

IL17RB 3.643 3.823 2.383 2.564 3.790 3.970

8 8

ITGA6 1.663 1.490 4.745 4.571 2.210 2.037

8

KDR 4.699 5.233 5.690 6.224 0.683 1.217 8 5 4 3

KIT 1.849 2.163 1.277 1.590 3.595 3.908

9

LCK 4.323 4.079 4.498 4.287 4.320 4.510

10

5 5

PROM1 8.036 7.889 8.058 7.911 4.978 4.830 1 1 2 2 3 4

PRSS16 4.019

4.179

3.555

PSIP1 3.532

3.522

3.506

SEMA4D 4.763 4.512 3.278 3.027 2.871 2.620 7 10

TNFRSF11A 4.984

4.670

3.826

5

9

7

TNFSF11 4.844 5.328 4.831 5.315 4.996 5.481 6 4 7 7 2 2

Table S2B. The down-regulated genes in undifferentiated stem cells compared to somatic cells.

Comparison (Fold Change) Ranking (Top 10)

Compare to PDPC

Compare to PFC

Compare to PGC

Compare to PDPC

Compare to PFC

Compare to PGC

GeneID NTU iCFB NTU iCFB NTU iCFB NTU iCFB NTU iCFB NTU iCFB

ANPEP -6.842 -6.513 -6.318 -5.989 -3.511 -3.182 3 5 8 10

C1R -3.625 -3.691 -6.097 -6.163 -3.529 -3.595

8

C1S -4.537 -4.640 -6.885 -6.987 -4.995 -5.097

5 5

CASP1 -3.911 -3.715 -5.301 -5.105 -6.092 -5.896

9 9

CCRL1 -5.441 -5.241 -8.535 -8.335 -3.099 -2.899

2 2

CD44 -6.022 -6.533 -6.267 -6.751 -5.134 -5.544 8 4 9 6

COLEC12 -5.030

-6.064

0.410

DPP4 -5.354

-5.856

-4.461

F3

-2.059

-1.289

-5.317

IFI16 -4.667 -4.514 -5.949 -5.802 -4.910 -4.708

IFI27 -6.220 -5.583 -3.113 -2.476 -6.303 -5.666 7 9

8 10

IFIT2 -4.697 -4.573 -6.052 -5.928 -4.947 -4.823

IFIT3 -7.093 -7.117 -7.139 -7.164 -6.419 -6.443 2 1 4 4 7 6

IGJ -2.989 -2.689 -1.722 -1.422 -6.423 -6.123

6 8

IL13RA1

-5.464

-5.572

-5.261

10

IL13RA2 -5.792 -5.929 -6.173 -6.310 -3.076 -3.213 9 8 10 7

IL1R1 -7.361 -6.950 -9.382 -8.972 -7.483 -7.072 1 2 1 1 3 3

IL6ST -5.230 -4.733 -6.483 -6.055 -5.839 -5.417

6 9

IL8 -0.048 -0.318 -3.844 -4.114 -8.158 -8.428

1 1

LGALS1 -5.555

-5.544

-5.577

10

LY96 -6.232 -6.092 -7.510 -7.370 -7.155 -7.015 6 7 3 3 4 4

NT5E -6.497 -6.120 -6.074 -5.791 -6.007 -5.639 5 6

OLR1

-0.166

-0.837

-5.296

PDGFRA -4.694 -4.139 -6.459 -5.904 -5.837 -5.282

7

SERPINE1 -5.235 -5.438 -3.360 -3.563 -6.719 -6.922

5 5

TGFB2 -4.703 -4.845 -2.791 -2.758 -6.010 -6.162

10 7

THBS1 -6.608 -6.850 -4.572 -4.814 -3.580 -3.822 4 3

VCAM1 0.202 0.601 -0.564 -0.166 -7.641 -7.243

2 2

Table S2C. The up-regulated genes in differentiated stem cells compared to somatic cells.

Comparison (Fold Change) Ranking (Top 10)

Compare to PDPC

Compare to PFC

Compare to PGC

Compare to PDPC

Compare to PFC

Compare to PGC

GeneID NTU iCFB NTU iCFB NTU iCFB NTU iCFB NTU iCFB NTU iCFB

ARG2 3.477 4.766 5.500 6.789 3.639 4.929

10 7 5

6

BMP7 4.916 4.773 4.386 4.243 4.704 4.561 9 9

6 7

CD24 4.488 4.080 8.368 8.345 4.228 3.900 10

1 1 9

CLU 4.136

-1.558

0.703

COLEC12 0.228 0.841 -0.806 -0.194 5.668 6.280

4 3

CXCL14 7.873 8.942 1.862 2.930 5.842 6.911 1 1

3 1

CXCR4 6.646 7.106 6.812 7.272 6.007 6.467 3 3 3 4 1 2

CYTL1 4.146

4.086

-1.268

EGR2 2.007

0.960

3.523

FGFR2 3.660 4.282 6.075 6.438 3.819 4.343

6 6 10 9

FGFR3 4.312 4.904 5.273 5.864 2.991 3.582

7 8 7

GATA3 6.473 5.559 6.775 5.861 5.890 4.976 4 5 4 8 2 5

IL11 4.135

3.705

1.853

KDR 5.444 6.948 6.435 7.939 1.428 2.931 6 4 5 2

LAMP3 3.346

3.225

3.551

MME -2.364 -1.211 -2.082 -0.929 4.812 5.964

5 4

NCAM1 1.877 2.011 4.959 5.093 0.495 0.629

NFATC2 3.915

4.267

3.856

OLR1 5.360 4.789 4.689 4.118 0.230 -0.341 7 8

PROM1 7.399 7.466 7.421 7.488 4.340 4.407 2 2 2 3 8 8

RNF128 5.502 4.077 4.993 3.567 -0.140 -1.565 5

TEK 4.269 5.268 4.428 5.427 2.802 3.801

6

10

THBD 2.013 2.583 5.212 5.430 3.714 4.083

9 9

10

TNFSF15 5.142

5.147

4.567

8

10

7

Table S2D. The down-regulated genes in differentiated stem cells compared to somatic cells.

Comparison (Fold Change) Ranking (Top 10)

Compare to PDPC

Compare to PFC Compare to

PGC Compare to

PDPC Compare to PFC

Compare to PGC

GeneID NTU iCFB NTU iCFB NTU iCFB NTU iCFB NTU iCFB NTU iCFB

ANPEP -6.724 -5.384 -6.200 -4.860 -3.393 -2.052 2 4 5 10

C1R -4.724 -3.400 -7.196 -5.872 -4.628 -3.304

3 4

C1S -4.513 -3.196 -6.861 -5.543 -4.970 -3.653

4 6

CASP1 -3.391 -2.833 -4.806 -4.333 -5.518 -5.007

8 7

CCL26 -2.162 -1.695 -0.768 -0.302 -5.123 -4.657

10 10

CCRL1 -5.733 -5.327 -8.827 -8.422 -3.391 -2.985 4 5 1 1

CD109 -4.851 -4.856 -4.584 -4.588 -1.782 -1.787 9 9

CD14 -4.839

-1.733

-3.021

10

CSTA -5.102 -4.832 -2.510 -2.240 -4.032 -3.762 6 10

CXCL1 -1.147

-4.535

-5.522

7

CXCL2 0.159 0.627 -3.857 -3.389 -5.400 -4.932

9 8

CXCL6 0.549 1.409 -5.114 -4.254 -7.750 -6.890

9

2 2

ICAM1 -4.560 -4.183 -3.662 -3.285 -5.025 -4.648

IFIT3 -4.822 -4.502 -4.869 -4.549 -4.148 -3.828

IGJ -2.436 -2.860 -1.168 -1.593 -5.869 -6.294

4 3

IL13RA2 -5.777 -5.889 -6.158 -6.270 -3.061 -3.173 3 3 6 3

IL1B -1.576

-1.977

-4.853

IL8 0.020 0.251 -3.776 -3.545 -8.090 -7.859

1 1

ITGA4 -4.618 -4.956 -4.067 -4.405 -3.452 -3.790

8

ITGB3

-3.935

-3.051

-4.904

9

LY96 -4.618 -4.200 -5.896 -5.479 -5.542 -5.124

7 7 6 5

MR1 -5.088 -4.965 -5.135 -5.012 -3.581 -3.458 8 7 8 9

MX1 -5.098 -5.209 -2.846 -2.957 -3.162 -3.273 7 6

PLXNC1 -3.599 -4.425 -4.721 -5.547 -0.013 -0.839

5

PSMB9 -4.287 -4.310 -5.058 -5.081 -5.067 -5.090

10 8

6

THBS1 -5.369 -5.999 -3.112 -3.963 -1.349 -2.971 5 2

THBS2 -6.780 -6.081 -7.349 -6.649 -6.370 -5.670 1 1 2 2 3 4

VCAM1 2.180 3.577 1.414 2.811 -5.663 -4.266

5

Table S3. The gene ontology pathway analysis indicated several significant pathways correlated with the 25 candidate immune genes.

GO_ID Term Number Of Genes p-value p-value_ fdr

p-value_ bonferroni

hsa04512 ECM-receptor interaction 16 4.67E-11 9.91E-09 9.91E-09

hsa04510 Focal adhesion 19 4.16E-08 4.41E-06 8.81E-06

hsa05146 Amoebiasis 12 2.23E-06 1.58E-04 4.73E-04

hsa04350 TGF-beta signaling pathway 8 5.70E-04 3.02E-02 1.21E-01

hsa05219 Bladder cancer 5 1.50E-03 6.34E-02 3.17E-01

hsa04060 Cytokine-cytokine receptor interaction 14 2.07E-03 7.31E-02 4.38E-01

hsa05144 Malaria 5 5.95E-03 1.80E-01 1.00E+00

hsa04610 Complement and coagulation cascades 6 1.04E-02 2.74E-01 1.00E+00

hsa04360 Axon guidance 7 1.85E-02 4.12E-01 1.00E+00

hsa05200 Pathways in cancer 13 1.94E-02 4.12E-01 1.00E+00

hsa05130 Pathogenic Escherichia coli infection 4 4.39E-02 8.16E-01 1.00E+00

Table S4A. Log ratio of the genes expression of undifferentiated pluripotent stem cells compared to somatic cells by Ingenuity Pathway Analysis.

Table S4B. Log ratio of the genes expression of differentiated pluripotent stem cells compared to somatic cells by Ingenuity Pathway Analysis.

PDPC: primary dermal papilla cells, PFC: primary foreskin fibroblast cells, PGC: primary granulosa cells. Human ES cells: NTU1 and human iPS cells: iCFB. *No significant.

NTU1 iCFB

Compare to PDPC

Compare to PGC

Compare to PFC

Compare to PDPC

Compare to PGC

Compare to PFC

GATA3 NS* NS NS NS NS NS

THBS4 2.33 2.10 2.58 2.72 2.49 2.97

IDO1 5.13 5.06 5.33 5.96 5.88 6.15

CR1L NS 1.91 2.15 3.46 3.77 4.01

TGFβ2 -4.10 -5.62 NS -4.46 -6.16 -3.72

TGFβ1I1 -5.52 -5.09 -4.88 -5.18 -5.02 -4.81

NTU1 iCFB

Compare to PDPC

Compare to PGC

Compare to PFC

Compare to PDPC

Compare to PGC

Compare to PFC

GATA3 6.63 6.09 6.71 5.98 5.44 6.07

THBS4 NS NS NS NS NS NS

IDO1 NS NS NS NS NS NS

CR1L NS NS NS NS NS NS

TGFβ2 NS NS NS NS NS NS

TGFβ1I1 NS NS NS NS NS NS

Table S5A. Up-regulated genes of undifferentiated pluripotent stem cells compare to somatic cells in IPA (Log 2 Ratio).

NTU1 iCFB

Compare to PDPC

Compare to PGC

Compare to PFC

Compare to PDPC

Compare to PGC

Compare to PFC

GATA3

RNF128 3.32

2.45 3.31

2.44

OLR1

ARG2 3.70 3.46 5.77 3.68 3.44 5.75

BMP7 2.70 2.60 2.93 2.79 2.69 3.02

IGF1R 1.78 2.22

2.58

EGR2

PILRB

1.18

EMB 2.31 2.05 4.55 2.40 2.14 4.64

CALCA 1.79 2.23 1.68 3.50 3.93 3.38

KIT 2.01 3.81

2.53 4.32

GPR83

TGFB2

THBS4 2.33 2.10 2.58 2.72 2.49 2.97

CALCB

3.56 3.85 3.74

CLEC4A

IDO1 5.13 5.06 5.33 5.96 5.88 6.15

HPRT1 2.22

2.16

1.46

CR1L

1.91 2.15 3.46 3.77 4.01

MIF

0.85

Table S5B. Down-regulated genes of undifferentiated pluripotent stem cells compare to somatic cells in IPA (Log 2 Ratio).

NTU1 iCFB

Compare to PDPC

Compare to PGC

Compare to PFC

Compare to PDPC

Compare to PGC

Compare to PFC

THBS2 -4.51 -3.92 -5.06 -4.61

-5.16

THBS1 -8.15 -5.48 -6.45 -8.09 -5.42 -6.39

LGALS1 -5.05 -4.96 -4.74 -4.38 -4.30 -4.08

CD14 -4.42

-4.86

CSTA -4.56

-4.43

HMOX1

-3.71

SERPINE1 -5.06 -6.41

-5.39 -6.75

ACTB

-3.19 -4.10 -4.32 -3.64

CD59 -3.89 -4.11 -4.22 -4.25 -4.48 -4.58

LGALS3 -4.01

-4.42

-4.01

IL1B

-4.59

-4.42

IL6

-3.96 -4.99

-4.52 -5.55

SOD3

-4.92

-4.95

CTSS

CCR1

CD274

IRF1

TGFB1I1 -5.25 -5.09 -4.88 -5.18 -5.02 -4.81

TGFB2 -4.10 -5.62

-4.46 -6.16 -3.72

OLR1

-4.79

-4.72

Table S5C. Up-regulated genes of 15 days differentiated pluripotent stem cells compare to somatic cells in IPA (Log 2 Ratio).

NTU1 iCFB

Compare to PDPC

Compare to PGC

Compare to PFC

Compare to PDPC

Compare to PGC

Compare to PFC

GATA3 6.63 6.09 6.71 5.98 5.44 6.07

RNF128 5.62

4.75 3.74

2.87

OLR1 4.20

4.27 3.69

3.76

ARG2 3.55 3.31 5.62 4.26 4.01 6.33

BMP7 3.34 3.24 3.57 2.93 2.83 3.16

IGF1R 2.58 3.02 1.67 2.43 2.86 1.51

EGR2

2.01

1.75

PILRB 1.37 1.53

1.31 1.46

EMB 1.48 1.22 3.72

3.26

CALCA

1.29

1.77 2.20 1.65

KIT

2.12

2.78

GPR83 1.34 1.65 1.29

1.46

TGFB2 2.28

2.76

1.57

THBS4

ALOX5AP

1.03

CALCB

2.76 3.05 2.94

CLEC4A

1.33

IDO1

HPRT1

CR1L

Table S5D. Down-regulated genes of 15 days differentiated pluripotent stem cells compare to somatic cells in IPA (Log 2 Ratio). Human ES cells: NTU1 and human iPS cells: iCFB

NTU1 iCFB

Compare to PDPC

Compare to PGC

Compare to PFC

Compare to PDPC

Compare to PGC

Compare to PFC

THBS2 -6.69 -6.10 -7.24 -7.53 -6.94 -8.08

THBS1 -4.95

-5.17

-3.35

LGALS1 -4.57 -4.49 -4.27 -3.35 -3.27 -3.05

CD14 -4.87 -2.81

-4.51

CSTA -4.83 -3.81

-4.45 -3.44

HMOX1 -4.20 -2.48 -4.12 -4.13

-4.06

SERPINE1 -3.02 -3.24

-3.34 -3.46

ACTB -3.42 -3.64 -2.95 -3.73 -3.95 -3.26

CD59 -2.31 -2.54 -2.64 -3.14 -3.37 -3.47

LGALS3 -2.16

-2.57

IL1B

-4.41

-4.38

IL6

-3.10 -4.13 -2.91 -4.17 -5.20

SOD3

-5.03

-4.82

CTSS

-3.02

-3.08

CCR1

-2.99

-3.00

CD274

-2.52

IRF1

-2.48

TGFB1I1

TGFB2

OLR1