Volume 15, Issue 11, Pages 2027-2036 (November 2007) Transgenic Enrichment of Cardiomyocytes From Human Embryonic Stem Cells  David Anderson, Tim Self, Ian R Mellor, Gareth Goh, Stephen J Hill, Chris Denning  Molecular Therapy  Volume 15, Issue 11, Pages 2027-2036 (November 2007) DOI: 10.1038/sj.mt.6300303 Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 1 Heterogeneity in human embryonic stem cell (hESC)-cardiomyocyte preparations. (a) Cells derived by dissociating beating HUES-7 embryoid bodies were stained with 4′,6-diamidino-2-phenylindole (blue, nuclei), Ki67 (green, proliferation) or α-actinin (red, cardiomyocytes). Note that a small proportion of the Ki67 positive cells also stain for α-actinin (arrows). Bar = 50 μm. (b) Proportion of Ki67 and α-actinin positive cells detected among 4,700 dissociated cells from HUES-7-derived beating areas across three independent experiments. Molecular Therapy 2007 15, 2027-2036DOI: (10.1038/sj.mt.6300303) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 2 Development of transgenic selection strategies. (a) Reverse transcriptase-polymerase chain reaction (PCR) analysis was carried out on RNA from undifferentiated HUES-7 cells (UD), embryoid bodies (EBs) from day 2 to 18 of differentiation or mechanically dissected beating EBs [cardiomyocyte (CM)]. (b) Quantification of MYH6 expression by Taqman PCR (relative to HPRT1). (c) Schematic representations of the plasmids used in this study. (Also see Materials and Methods.) (d) Quantification of percentage of green fluorescent protein (GFP) positive HUES-7 cells by fluorescence-activated cell sorting analysis 48 hours after transfection with pCAG-GFP-IRES-PAC. Lanes shown are C1, no lipid/no DNA control; C2, no lipid + DNA control; 1, Lipid only control; 2–4 show lipid, DNA ratios of 3:1, 2:1 and 1:1 respectively; E, electroporation. Lipid transfection reagents were GJ, GeneJammer; M, Metafectene; F, Fugene; Ex, ExGen 500; G, GeneJuice; L, Lipofectamine 2000. Data were from three independent experiments, each performed in duplicate. (e) PCR analysis was carried out using DNA from 1, HUES-7; 2, HUES-7GIP; 3, HUES-7TK; 4, HUES-7F; 5, HUES-7R. IP3R3 product is 1,257 base pair (bp); this was used as a control to demonstrate amplification from genomic DNA rather than RNA, which gives a product of 215 bp. IRES, internal ribosome entry site; αMHC, αmyosin heavy chain; PAC, puromycin-N-acetyltransferase. Molecular Therapy 2007 15, 2027-2036DOI: (10.1038/sj.mt.6300303) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 3 Phenotypic comparison of parental and transgenic HUES-7 lines. (a) Brightfield images of morphology and (b) immunofluorescence images of OCT4 expression. (c) Fluorescence-activated cell sorting plots showing SSEA-4 (y-axis) and green fluorescent protein (GFP) (x-axis) expression. The lower left quadrant indicated by the dotted lines gated 95% of HUES-7 cells to account for non-specific secondary antibody binding and autofluorescence. (d) Representative images of embryoid bodies (EBs) on day 4 of differentiation after formation by forced aggregation. Numbers represent mean ± SEM EB size (μm) from a minimum of 80 EBs measured from at least four independent experiments. Bar = 100 μm. (e) The percentage of beating EBs assessed per line was from between 8 and 21 independent experiments (representing 768–2,016 EBs). Molecular Therapy 2007 15, 2027-2036DOI: (10.1038/sj.mt.6300303) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 4 Micro Electrode Array analysis of beating embryoid bodies (EBs). (a) The micro electrode array (MEA) comprises 60 contacts (Ai) that connect a preamplifier to a grid of electrodes spaced at 200 μm intervals (arrow in Ai and Aii). Bar = 200 μm. Beating EBs were seeded to the MEA (Aiii) and extracellular field potentials were recorded at rest (basal) or after treatment with isoprenaline, propranolol or isoprenaline + propranolol (b; recordings shown are with 1,000 nmol/l isoprenaline). ISI, interspike interval used to calculate beats per minute (bpm); FPmax, peak to peak amplitude; FPmin, minimum potential. For each line basal ISI (c) or beat rates (d) between the lines was similar. (e) Responses of lines plotted relative to basal after treatment with 1–1,000 nmol/l isoprenaline (±100 nmol/l propranolol). HUES-7 (squares), HUES-7GIP (triangles), HUES-7TK (open squares), HUES-7F (diamonds), HUES-7R (open triangles). As data for FPmax and FPmin were comparable, only FPmax are shown. Molecular Therapy 2007 15, 2027-2036DOI: (10.1038/sj.mt.6300303) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 5 Assessment of transgene expression. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was carried out using RNA from (a) undifferentiated cells or (b) beating embryoid bodies (EBs) from HUES-7 (1), HUES-7GIP (2), HUES-7TK (3), HUES-7F (4) and HUES-7R (5). (c) RT-PCR from undifferentiated HUES-7R cells (UD), EBs from day 4 to 16 of differentiation and beating EBs [cardiomyocyte (CM)] Brightfield and fluorescence images of (d) HUES-7GIP undifferentiated cells or (e) beating EBs. Bar = 100 μm. Cultures of (f) undifferentiated or (g) differentiated HUES-7 and HUES-7TK cells were treated with ganciclovir (GCV) for 5 days. Viable cell counts were from two independent experiments, each performed in duplicate. HUES-7 (solid squares) and HUES-7TK (open squares). Molecular Therapy 2007 15, 2027-2036DOI: (10.1038/sj.mt.6300303) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 6 Transgenic enrichment of human embryonic stem cell (hESC)-cardiomyocytes. (a) Beat rates of single cardiomyocytes from dissociated beating embryoid bodies (EBs). H, HUES-7; GIP, HUES-7GIP; TK, HUES-7TK; F, HUES-7F; R, HUES-7R. (b) Beat rates of unselected or drug-selected single cardiomyocytes in the absence (white bars) or presence (black bars) of 1,000 nmol/l isoprenaline. (c) An assessment of the action potentials following whole-cell patch-clamp electrophysiology was used for identifying cardiomyocyte subtypes. (d) Dissociated beating HUES-7TK EBs were untreated or treated with 10 mmol/l ganciclovir (GCV) for 7 days before staining with 4′,6-diamidino-2-phenylindole (DAPI), (blue, nuclei), Ki67 (green, proliferation) or α-actinin (red, cardiomyocytes). The dotted ellipse and arrows indicate cells that are negative for α-actinin. (e) Quantification of Ki67 and α-actinin positive cells within untreated (open bars) or GCV treated (solid bars) HUES-7TK cultures. Data are Mean ± SEM from three experiments (total of 10,144 nuclei scored). (f) Images are from dissociated beating HUES-7R EBs untreated or treated with 100–200 ng/ml puro for 3, 5, or 7 d before staining with DAPI or α-actinin. (g) Quantification of α-actinin positive cells within HUES-7F (open bars) or HUES-7R (solid bars) with or without puro treatment. Data are mean ± SEM from 2 (HUES-7F; total of 7,328 nuclei scored) or 5 (HUES-7R; total of 10,077 nuclei scored) experiments. (h) Reverse transcriptase-polymerase chain reaction analysis was carried out on RNA from unselected beating EBs (US) or puromycin selected cardiomyocytes (PS). CMs, cardiomyocytes. Bar = 50 μm. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Molecular Therapy 2007 15, 2027-2036DOI: (10.1038/sj.mt.6300303) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions