Functional Mesenchymal Stem Cells Derived From Human Induced Pluripotent Stem Cells Attenuate Limb Ischemia in Mice by Qizhou Lian, Yuelin Zhang, Jinqiu Zhang, Hua Kun Zhang, Xingang Wu, Yang Zhang, Francis Fu-Yuen Lam, Sarang Kang, Jian Chuan Xia, Wing-Hong Lai, Ka-Wing Au, Yen Yen Chow, Chung-Wah Siu, Chuen-Neng Lee, and Hung-Fat Tse Circulation Volume 121(9):1113-1123 March 9, 2010 Copyright © American Heart Association, Inc. All rights reserved.

Figure 1. Generation of single-cell level–derived MSCs from differentiating human iPSCs. Figure 1. Generation of single-cell level–derived MSCs from differentiating human iPSCs. A, The typical morphology of human iPSC-MSCs. B, Immunostaining for CD24 and CD105 in human iPSCs and iPSC-MSCs (i through viii). C, Limiting dilution of CD24−/CD105+ MSCs formed fibroblast-like colony-forming units (i through iii) and a representative GFP+ MSC colony beginning from a single-cell level (iv through vi). D, Western blotting for pluripotency-associated proteins Oct4, Nanog, and Sox2 in the iPSC lines iPSC(iMR90)-MSC10 and iPSC(foreskin)-MSC11. E, Immunostaining of Oct4 in iPSCs and iPSC-MSCs. F, PCR for mouse DNA repeat sequences c-mos and human DNA repeat sequences Alu-sx among iPSC-MSCs, mouse fibroblast feeder (MEF), and iPSC on MEF. G, Surface antigen profiling by FACS in iPSC-MSC cultures for CD44, CD49a, CD49e, CD73, CD105, CD166, CD34, CD45, CD133, and TRA-81. Qizhou Lian et al. Circulation. 2010;121:1113-1123 Copyright © American Heart Association, Inc. All rights reserved.

Figure 2. Characterization of iPSC-MSCs. Figure 2. Characterization of iPSC-MSCs. A, Oil Red staining for adipogenesis (i); Alizarin Red staining for osteogenesis (ii); Alcian Blue staining for chondrogenesis (iii); and immunoreactivity for collagen type II (iv). B, Transcription levels of adipocyte genes PPARG2 and LPL, ostoecyte genes Osteocalcin and ALP, and chondrocyte genes Sox9 and AGC were measured by RT-PCR. GADPH was used as loading control. C, Surface antigen profiling in iPSC-MSCs at 120 population doublings for CD44, CD49a, CD73, CD90, CD105, CD106, CD34, CD45, and CD133. D, Methylation status of Oct4 promoter among iMR90, iPSC(iMR90)-5, and iPSC(iMR90)-MSC. E, The 3-group comparison showed a significant difference in telomerase activity among BM-MSCs, iPSC(iMR90)-MSC10, and iMR90 (P<0.001). *P=0.02, **P<0.001 vs iPSC-MSCs; n=3 in each group. Qizhou Lian et al. Circulation. 2010;121:1113-1123 Copyright © American Heart Association, Inc. All rights reserved.

Figure 3. Transplantation of iPSC-MSCs attenuates hind-limb ischemia. Figure 3. Transplantation of iPSC-MSCs attenuates hind-limb ischemia. A, Representative photos of vehicle (DMEM)-, BM-MSC–, or iPSC-MSC–treated animals at days 0 (after surgery), 7, and 21. B, At day 21 after treatment, the 3-group comparison showed a significant difference in physiological status of ischemic limbs rated in 3 levels: limb salvage, foot necrosis, and limb loss. P=0.0003; n=15 per group. C, At day 21 after transplantation, hematoxylin and eosin staining for ischemic limbs showed massive muscle degeneration with replacement by infiltration of numerous granulocytes and neutrophils in the vehicle group. In the BM-MSC and iPSC-MSC groups, the muscle degeneration of limb ischemia was largely protected. Masson trichrome staining that showed fibrosis was obviously attenuated in iPSC-MSC–treated limbs. Qizhou Lian et al. Circulation. 2010;121:1113-1123 Copyright © American Heart Association, Inc. All rights reserved.

Figure 4. Improvement in blood flow and limb functions after cell transplantation. Figure 4. Improvement in blood flow and limb functions after cell transplantation. A, Representative laser Doppler flow imaging showed dynamic changes in blood perfusion in limb ischemia at days 0, 14, and 21. B, The improvement in blood perfusion was significantly different in the 3-group comparison (P<0.001; n=15 per group). Blood perfusion was not significantly changed in the vehicle group but gradually recovered in the BM-MSC and iPSC-MSC groups over 21 days (*P<0.001 vs day 0). The iPSC-MSC group was significantly higher than the BM-MSC group in blood perfusion at both day 14 (#P=0.02) and day 21 (#P=0.01). The 3-group comparison showed a significant difference in scores of ambulatory impairment (ii; P<0.001; n=15 per group) and ischemic tissue damage (iii; P<0.001; n=15 per group). Compared with the vehicle group, scores of ambulatory impairment (ii) and tissue damage (iii) were significantly reduced in the BM-MSC and iPSC-MSC groups at day 21 (*P<0.001). Compared with the BM-MSC group, significantly lower ambulatory impairment and tissue damage scores were observed in iPSC-MSC–treated mice at day 21 (iii; #P<0.001). Qizhou Lian et al. Circulation. 2010;121:1113-1123 Copyright © American Heart Association, Inc. All rights reserved.

Figure 5. Differential effects of iPSC-MSCs and BM-MSCs on hind-limb ischemia. Figure 5. Differential effects of iPSC-MSCs and BM-MSCs on hind-limb ischemia. A, In a mouse ESC-induced teratoma with a mixture of iPSC-MSCs, differentiation of iPSC-MSCs was verified by immunostaining of human desmin and CD31. B, Immunostaining of human CD31 (i and iii, arrows) and quantitative analysis by FACS (ii and iv) after cells were exposed to endothelial differentiation conditions for 5 days. C, Immunostaining of human α-SMA (i and iii, arrows) and quantitative analysis by FACS (ii and iv) after cells were exposed to smooth muscle differentiation conditions for 5 days (*P=0.038; n=3). D, Representative imagines of fibrosis with Masson trichrome stain (i through iii), inflammation with CD45+ stain (v through vii), myogenesis with desmin plus stain (ix through xi), smooth muscle differentiation with α-SMA+ stain (xiii through xv), and endothelial differentiation with CD31+ stain (xvii through xix) on cross sections in the vehicle, BM-MSC, or iPSC-MSC group. Quantitative measurement was expressed as percent of positive staining vs total per muscle area. The 3-group comparison of fibrosis, inflammation, myogenesis, α-SMA+, and CD31+ staining was significantly different (*P<0.001 vs vehicle group; #P<0.001 vs BM-MSCs). iv:*P<0.001, #P=0.002; viii:*P=0.003, #P=0.02; xii: *P<0.001, #P=0.03; xvi: *P<0.001, #P=0.02; xx: *P<0.001. n=6 per group. Qizhou Lian et al. Circulation. 2010;121:1113-1123 Copyright © American Heart Association, Inc. All rights reserved.

Figure 6. Cell retention and paracrine factors. Figure 6. Cell retention and paracrine factors. A, Immunostaining of antihuman α-SMA of ischemic limb tissue in the iPSC-MSC and BM-MSC groups at days 7, 21, and 35 after cell transplantation (i through vi, arrowheads show small arteries). B, Reverse-transcription PCR for human α-SMA in ischemic limb tissues retrieved 7 to 35 days after transplantation (i). C, Representative photographs showing that pLL3.7-GFP labeled iPSC-MSCs were immunoreactive for human CD31 in ischemic limb tissues at day 35 after transplantation (ii). Immunostaining of human nuclear antigen (HNA) in iPSC-MSC– and BM-MSC–treated tissues at day 35 after transplantation (iii). D, Comparison of human stromal derived factor (SDF)-1α (*P=0.04), stem cell factor (SCF; *P=0.03), bFGF (*P=0.03), hepatocyte growth factor (HGF; *P=0.008), β-nerve growth factor (b-NGF; *P=0.01), and vascular endothelial growth factor (VEGF) contents in supernatants between iPSC-MSCs and BM-MSCs exposed to hypoxia (5% O2) for 48 hours (n=3 per group). E, Tubular formation of iPSC-MSCs on Matrigel Matrix under 1% FBS culture medium and supernatants of iPSC-MSCs or BM-MSCs (i). The 3-group comparison showed a significant difference (P<0.001) by quantitative analysis of tubular branches of iPSC-MSCs in culture medium of iPSC-MSCs, BM-MSCs, or 1% FBS (ii). *P<0.001 vs vehicle group; n=3. Qizhou Lian et al. Circulation. 2010;121:1113-1123 Copyright © American Heart Association, Inc. All rights reserved.