hipscs were derived from human skin fibroblasts (CRL-2097) by ectopic

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1 Generation and characterization of hipscs hipscs were derived from human skin fibroblasts (CRL-2097) by ectopic expression of OCT4, SOX2, KLF4, and C-MYC as previously described 1. hipscs showed typical hesc morphology, and expressed stem cell markers such as SSEA-4, OCT4, AP staining, TRA-1-60, and NANOG (Figure S2A). Ectopic genes were not expressed in hipscs (Figure S2B). And the karyotype indicates that the number and morphology of the chromosomes are normal, 44+XY (Figure S2C). Promoter regions of the pluripotent genes were unmethylated in hipscs, like in hescs (Figure S2D). This result indicates that the hipscs used in this study were epigenetically reprogrammed. Western blotting To examine the activation status of the MEK/ERK and BMP4 signaling pathways, we monitored the phosphorylation of ERK1/2 and SMAD1/5/8 in hescs treated with PD98059 or BMP4. For the isolation of total proteins, hescs were lysed in the PRO- PREP TM protein extraction solution (Intronbio, Seongnam, Korea), and the extracted proteins were quantified by Bradford assay. Total proteins (20 µg) were separated by 8% SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad). The blotted membranes were blocked with 5% skim milk in Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBST; 10 mm Tris-HCl, ph 7.5, 150 nm NaCl, and 0.1% Tween-20). The membranes were then incubated at 4 overnight with primary antibodies for Oct3/4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:1000), T (R&D Systems, 1:500), ERK1/2, p- ERK1/2, p-smad1/5/8 (Cell Signaling Technologies, 1:1000), SMAD1 (Abcam, 1:1000),

2 or α-tubulin (Sigma, 1:3000; loading control). After being washed with TBST, the samples were treated with the secondary antibody in blocking solution at 4 for 1 h. The membrane was then washed and signals were detected using an ECL system (Pierce, Rockford, IL, USA) as recommended by the manufacturer s protocol. Analysis of DNA methylation by a bisulfite sequencing method Analysis of DNA methylation at CpG sites on the promoters of OCT4, REX1, and NANOG genes was performed as previously described 2. Briefly, genomic DNA was subjected to sodium bisulfite treatment using the EZ DNA Methylation-Gold Kit according to the protocol of the manufacturer (Zymo Research, Orange, Calif). Then, bisulfate-treated DNA (25 to 50 ng) was amplified by PCR. The amplified PCR products were purified by using AccuPrep plasmid Mini extraction Kit (Bioneer, Korea), and subcloned into pgem-t EASY vector (Promega, Madison, WI, USA). For each cell type, 6 clones were sequenced by using M13 primer, and were further analyzed by using a web-based program (Blast-2) or software (BiQ Analyzer). Analysis of near infrared fluorescence imaging For time-series ICG imaging, mice under ketamine-xylazine anesthesia were injected with an intravenous bolus injection of ICG (0.1 ml of 400 μmol/l; Sigma) into the tail vein. ICG fluorescence images were obtained for 12 min in 1-s intervals immediately after injection. In regions of interest (ROI) corresponding to limbs in the acquired timeseries NIRF imaging, the perfusion rates of pixels (typically around ) in the ROI were analyzed by using a C ++ -based analysis program according to a mathematical model to calculate the regional (pixel) perfusion rate and visualized as a pseudocolor-coded perfusion map. The perfusion rate (%/min) was defined as the fraction of blood exchanged per min in the vascular volume of ROI. The probability of necrosis of ischemic limbs was

3 also estimated. Briefly, to determine the probability of necrosis, regional perfusion rates of an ischemic limb were measured at 2 h after surgery, and necrotic regions in the ischemic limbs were assessed on POD 7. Necrosis probability of the ischemic tissue was measured using the correlation between the regional perfusion rate and the necrosis of the corresponding region. Necrosis probabilities of about regions from 9 mice injected with hesc-derived CD34 + cells, about regions from 9 mice with EGM-2 medium, and about regions from 5 mice with hesc-derived CD34 - cells were averaged, respectively. Necrosis probabilities of ischemic hindlimbs were classified into 3 groups; poor perfusion rate (lower than 15 %/min), moderate perfusion rate (16 to 120 %/min), and good perfusion rate (higher than 120 %/min). 1. Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131: Lee TH, Song SH, Kim KL, et al. Functional recapitulation of smooth muscle cells via induced pluripotent stem cells from human aortic smooth muscle cells. Circ Res. 2010;106:

4 Table S1. Primer lists used in this study

5 Table S2. Primers specific for human angiogenic genes Table S3. Primers specific for mouse angiogenic genes

6 Figure S1

7 Figure S1. Characteriztion of CHA4-hES cells. A. Expression of ES-specific markers such alkaline phosphatase, OCT4, SSEA-4, TRA-1-60, and TRA B. Normal G-banded karyotype of CHA4-hESCs. C. Various cell types in teratomas derived from CHA4-hES. a. Epithelium (ectoderm). b. Neurons (ectoderm). c. Neural rosettes (ectoderm). d. Muscle fiber (mesoderm). e. Cartilage (mesoderm). f. Gut epithelium (endoderm).

8 Figure S2

9 Figure S2. Characterization of ipscs derived from human fibroblasts. A. Morphology, alkaline phosphatase staining, and immunostaining of stem cell markers (SSEA4, OCT4, NANOG, and TRA-1-60) in hipscs. B. RT-PCR for OCT4, SOX2, KLF4 and C-MYC in hipscs. C. Normal G-banded karyotype of hipscs. D. Three germ layer tissues (ectoderm, mesoderm, and endoderm) in teratoma derived from hipscs. a. Neural rosette. b. Cartilage. c. Gut-like epithelium. E. DNA methylation patterns of OCT4, REX1, and NANOG promoters in hipscs, H9 hescs, and fibroblasts. Open and filled circles indicate unmethylated and methylated CpG dinucleotides, respectively. Methyaltion level of each promoter region was calculated as methylated CpG sites per total CpG sites.

10 Figure S3. Overall procedure for differentiating hescs and hipscs to vascular and hematopoietic lineages. It consists of four phases, as follows. I: Mesoderm induction stage. hescs and hipscs were incubated in ESCM supplemented with 50 µm of PD98059 (Promega, Madison, WI) and ng/ml of BMP4 (Peprotech, Rocky Hill, NJ) for 3 and 5 d, respectively. II: Differentiation of mesodermal cells to CD34 + cells. The ESCM containing PD98059 and BMP4 was changed to ESCM supplemented with 100 ng/ml of VEGF-A and 50 ng/ml of bfgf. III: CD34 + MACS sorting. At 6~9 d of culture, CD34 + cells were isolated using CD34 + microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany). IV: Terminal differentiation stage. IV-1: Differentiation of CD34 + cells to endothelial cells. CD34 + cells were cultured in EGM-2MV (CAMBREX, Rutherford, NJ) supplemented with 100 ng/ml of VEGF-A and 50 ng/ml of bfgf (R&D Systems, Minneapolis, MN) for 12 d. IV-2: Differentiation of CD34 + cells to smooth muscle cells. For differentiation into smooth muscle cells, the CD34 + sorted cells were cultured in EGM-2 supplemented with 100 ng/ml of PDGF-BB and 50 ng/ml of bfgf for d. IV-3: Differentiation of CD34 + cells to hematopoietic cells. The sorted CD34 + cells were placed on MethoCult GF H4434 (StemCell Technologies, Vancouver, BC, Canada) and incubated at

11 37 in a 5% CO 2 -containing atmosphere for 3 weeks. The presence of hematopoietic cells was confirmed by a CFU (Colony Forming Unit) assay.

12 Figure S4. Expression of mesoderm marker T in PDB4-treated hipscs. hipscs were cultured on Matrigel for feeder-free culture and treated with 50uM of PD98059 and 10 ng/ml of BMP4 for 5 d. Expression of pluripotency (Tra-1-81, alkaline phosphatase, and SSEA-4) and mesoderm (T) markers was observed under the fluorescence microscope after immunostaining. Scale bar is 200 µm.

13 Figure S5. Expression of hematopoietic/endothelial progenitor marker genes in PDB4- treated hescs cultured in ESCM supplemented with Vb. Hematopoietic/endothelial progenitor marker genes (RUNX1, KDR, VE-CADHERIN, and CD34) were highly expressed in PDB4-treated hescs cultured in ESCM supplemented with 100 ng/ml of VEGF-A and 50 ng/ml of bfgf.

14 Figure S6

15 Figure S6. Therapeutic effects of hipsc-derived CD34 + cells on neovasculogenesis in ischemic hindlimb mice. A. A perfusion map. B. Detection of direct (top, yellow arrowhead) and indirect(button, red arrowhead) contributions of hipsc-derived CD31 + cells in ischemic hindlimbs. Scale bar is 20μm. C. Comparison of necrosis probability in ischemic hindlimbs between hesc- and hipsc-derived CD31 + cells.

16 Figure S7.Flow cytometric analyses of the hesc-derived-cd34 + cells. A. Expressions of CD31, CD73, and CD90 in isolated hesc-derived CD34 + cells. B. Low expression of CD43 (Leukosialin) in hesc-derived CD34 + cells.

17 Figure S8. Low differentiation potential of hipsc-derived CD34 + cells to hematopoietic lineage. A, CFU assay in hesc- (left) and hipsc-derived CD34 + cells (right). Scale bar is 200μm. B. Expression of hematopoietic genes in hesc- and hipsc-derivatives. Hematopoietic cells are abbreviated as HPCs.