File S1 - Bioluminescent Imaging of Genetically Selected Induced Pluripotent Stem Cell-Derived Cardiomyocytes after Transplantation into Infarcted Heart of Syngeneic Recipients

Figure S1, Targeted integration of a firefly luciferase reporter gene into the ROSA26 locus. Structures of targeting vector, wild-type ROSA26 locus and ZFN-targeted ROSA26 locus are shown. Targeting vector pDonor-pUbC [luc2/Hygro]-ROSA26 was used for insertion of the transgene cassette into the ROSA26 locus by homology-directed repair of the double-strand brake induced by ZFN encoded by plasmids pCMV-RosaL6 and pCMV-RosaR4. The targeting construct contains the UbC promoter driving constitutive expression of FLuc gene and SV40-promoter driving expression of a selectable marker for hygromycin resistance flanked by the left (796 nt) and right (815 nt) homology arms. The structure of the wild-type ROSA26 locus depicts the ZFN^Rosa cleavage site within an intronic XbaI site (arrowhead). Small open boxes indicate exon regions. The location of EcoRI sites and Southern blot probe located in the ROSA26 locus upstream of the transgene integration site are indicated. The sizes of EcoRI restriction fragments of wild-type allele and targeted ROSA26 allele that can be detected with the Southern blot probe are 15630 nt and 4066 nt, respectively. Forward PCR primer (F) binding to targeting vector and reverse primer (R) binding to ROSA26 locus downstream of the integration site were used to amplify a 950 nt diagnostic fragment and are indicated by black arrowheads. Figure S2, FLuc-αPIG-iPSC-derived CM are structurally and functionally intact. A. Flow cytometric analysis of puromycin selected FLuc-αPIG -iPS-CM (clone # C3) at day 16 of differentiation. Left panel: gated cell population (2×10⁴ events) on forward and sideward scatter dot plot. Right panel: dot plot showing the distribution of propidium iodide (PI)-positive cells and EGFP-positive viable iPS-CM in the gated cell population. B. Semiquantitative RT-PCR analysis for indicated cardiac specific transcripts in purified FLuc-αPIG-iPS-CM (clone # C3, upper panel) and parental iPSC-derived CM (lower panel). C. Immunofluorescence detection of EGFP, α-actinin 2 and cardiac troponin T (cTnT) in purified FLuc-αPIG -iPS-CM (clone # C3) plated on fibronectin-coated dishes. Nuclei were stained with DAPI. Scale bar: 20 µm, magnifications were enhanced 2.15-fold. D. Representative action potentials (AP) of spontaneously beating FLuc-αPIG-iPS-CM recorded by the whole-cell current-clamp technique before, during and after wash-out (WO) addition of 1 µM isoproterenol (Iso) and 1 µM carbachol (Cch). E. Quantitative analysis of beating rates of FLuc-αPIG-iPS-CM before, during and after (wash-out, WO) addition of Iso or Cch. Data are given as mean ± SD (n = 5 cells in each group; **p<0.01; ***p<0.001). Figure S3, Generation and characterization of Fluc-activity in pPGK-FLuc αPIG-ESC lines. A. Schematic representation of the pGL4.14-pPGK [luc2/Hygro] plasmid used to generate transgenic pPGK-FLuc-αPIG-ESC lines. B. Bioluminescence signal intensity (BLI) in 17 pPGK-FLuc ESC clones obtained after hygromycin selection. Values represent relative luminescence units (RLU) in live cell measurements of 1×10⁶ cells/well in a single experiment. C. Linear relationship between cell dose and BLI of pPGK-FLuc ESC clone F2. Data are given as mean ± SD of triplicate measurements. D. Four pPGK-FLuc-αPIG-ESC clones with highest FLuc-activity were subjected to spontaneous in vitro differentiation. BLI was measured in undifferentiated ESC, cells from dissociated day 6 and day 16 EB and in puromycin-selected cardiomyocytes on day 16 of differentiation (d16 CM) in the GENios Pro microplate reader. Data are given for a single measurement of 5×10⁵ cells/well of a 96-well plate. E. Relative FLuc transcript levels in undifferentiated ESC, day 16 EB and purified day 16 cardiomyocytes (d16 CM) of pPGK-FLuc ESC clones B2 and B5 as determined by RT-qPCR. Note the dramatic reduction of FLuc mRNA expression in differentiating EB cells and purified CM. Data were normalized to GAPDH as a housekeeping gene control and are given as mean ± SD of triplicate measurements. Figure S4, Significant decline of the bioluminescence signal intensity on the first day after transplantation of FLuc-expressing iPSC-derived cardiomyocytes. 5×10⁵ purified pUbC-FLuc iPS-CM or pUbC-FLuc-ROSA iPS-CM were transplanted into the cryoinjured heart of syngeneic mice (n = 4) and BL measurements were performed on day 0, day 1 and day 3 after cell injection. Data are shown relative to BL signal intensity that was determined on day 0 six hours after CM transplantation. Statistical analysis of day 0 versus day 1 and day 1 versus day 3 BL intensities was performed using the two-tailed paired Student's t-test. p-values for pUbC-FLuc-ROSA and pUbC-FLuc iPS-CM are shown on upper and lower lines, respectively. The pattern of BL fluctuation in mice injected with pUbC-FLuc-ROSA iPS-CM was similar to that observed in pUbC-FLuc iPS-CM-transplanted mice but did not reach statistical significance due to a large coefficient of variation within the former group on day 0 and day 3. Figure S5, No significant effect on capillary density and fibrotic area four weeks after transplantation of iPS-CM into cryoinjured hearts. A. Scar size was evaluated by Masson's trichrome (MTC) staining of histological sections of hearts four weeks after cryoinfarction and injection of either PBS saline (sham group) or iPS-CM. n = 5 animals for both groups, p = 0.118. B. Representative images of MTC stained sections of sham and iPS-CM treated hearts. C. Capillarization in periinfarct region of sham and iPS-CM-transplanted hearts was assessed by calveolin staining (green fluorescence). n = 5 animals for both groups, p = 0.0832. D. Representative calveolin stained sections of sham and iPS-CM treated hearts. Scale bars: 50 µm. Selected areas in panels B and D are shown magnified in the corresponding right hand panels.

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