Transgenic Enrichment of Cardiomyocytes From Human Embryonic Stem Cells
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Transgenic Enrichment of Cardiomyocytes From Human Embryonic Stem Cells David Anderson1,2, Tim Self3, Ian R Mellor4, Gareth Goh1,5, Stephen J Hill3 and Chris Denning1 Wolfson Centre for Stem Cells, Tissue Engineering and Modelling, University of Nottingham, Nottingham, UK; 2Institute of Genetics, University of Nottingham, Nottingham, UK; 3Institute of Cell Signalling, University of Nottingham, Nottingham, UK; 4School of Biology, University of Nottingham, Nottingham, UK; 5Division of Therapeutics and Molecular Medicine, University of Nottingham, Nottingham, UK 1
To realize the full scientific and clinical potential of human embryonic stem cell (hESC)-cardiomyocytes, stra tegies to overcome the high degree of heterogeneity of differentiated populations are required. Here we demonstrate the utility of two transgenic approaches in enrichment of cardiomyocytes derived from HUES-7 cells: (i) negative selection of proliferating cells with the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/ GCV) suicide gene system; and (ii) positive selection of cardiomyocytes expressing a bicistronic reporter [green fluorescent protein (GFP)-internal ribosome entry site (IRES)-puromycin-N-acetyltransferase (PAC)] from the human αmyosin heavy chain promoter. Parental and transgenic HUES-7 cells were similar with regard to morphology, pluripotency marker expression, differentiation, and cardiomyocyte electrophysiology. Whereas immunostaining of dissociated cardiomyocyte preparations expressing HSVtk or PAC contained 10 nmol/l (Figure 4e; Supplementary Table S1). There was no significant difference for the levels of response between the lines at specific doses (Figure 4e; Supplementary Table S1). With the exception of 1,000 nmol/l concentration, the effect of the isoprenaline was negated by co-incubation of beating areas from all lines with propranolol (Figure 4e; Supplementary Table S1). Interestingly, isoprenaline or propranolol + isoprenaline treatment regimes did not elicit any significant changes in FPmax or FPmin in any of the lines (Figure 4e and data not shown). Thus, taken together these results indicate that there is a high degree of phenotypic similarity between the transgenic lines in the undifferentiated state, while differentiating, or in the beating clusters formed.
Transgene expression and cardiomyocyte enrichment We observed appropriate expression of GFP, HSVtk and Neo after RT-PCR analysis of RNA from undifferentiated and differentiated HUES-7GFP, HUES-7TK and HUES-7R (Figure 5a and b). The onset of GFP and endogenous MYH6 expression was also similar in differentiating HUES-7R cells (Figure 5c). However, while appropriate transcripts were detected in undifferentiated HUES-7F cells (Figure 5a), GFP expression was not detected in the derived beating EBs (Figure 5b). At the protein level, strong Molecular Therapy vol. 15 no. 11 nov. 2007
GFP fluorescence was evident in undifferentiated HUES-7GIP cells (Figure 5d) and was retained after differentiation in beating clusters (Figure 5e). Functional transgene expression was also observed in HUES-7TK. While the dose of GCV required to kill 50% (IC50) of untransfected, undifferentiated HUES-7 cells over a 5 day treatment period was 27 µmol/l, this reduced to 8.5 nmol/l in undifferentiated HUES-7TK cells, indicating a shift in sensitivity of >3 logs (Figure 5f). Challenge of monolayer cultures established from EBs on day 25 of differentiation with GCV for 5 days showed an IC50 of >100 µmol/l for HUES-7 cells, which reduced to 46 nmol/l in HUES-7TK cells, representing a minimum 220-fold difference (Figure 5g). To determine whether drug treatment of HUES-7TK or HUES-7F/R could lead to enrichment of cardiomyocytes, beating EBs from each transgenic line were disaggregated to single cells on day 25 of differentiation. Between the lines, single spontaneously contracting cardiomyocytes showed similar beat rates (P = 0.57; one-way ANOVA; Figure 6a). Cultures of HUES-7TK or HUES-7F and HUES-7R were then incubated for up to 10 days with or without 10 µmol/l GCV or 100–200 ng/ml puromycin, respectively. Cardiomyocytes continued to beat for up to 6 weeks (the longest period studied) after selection. Beat rate (P = 0.9; one-way ANOVA; Figure 6b) and positive chronotropic response to isoprenaline (P = 0.82; one-way ANOVA; Figure 6b) of unselected cardiomyocytes was similar to those receiving selection. In addition, whole-cell patch-clamp analysis showed action potential characteristics (Figure 6c30) were 2031
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Beat rate (bpm)
60 40 20 H
Ventricular Atrial Pacemaker H
− + − + − + + + Treatment/reactivity
− + − −
70 60 50 40 30 20 10 0 GCV �-actinin Ki67 % Cells relative to total nuclei
120 100 80 60 40 20 0
% CMS relative of total nuclei
Beat rate (bpm)
− + + −
3 5 Days in puromycin
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.
c omparable for parental HUES-7, GCV selected HUES-7TK and puromycin selected HUES-7R cardiomyocytes. ‘Ventricularlike’ and ‘non-ventricular-like’ morphologies could be detected from all lines; the latter constitutes atrial- and pacemaker-like morphologies because in most cases these could not be distinguished. Interestingly, the association of ‘ventricular-like’ cells with MYH6 expression differs from reports on mouse ESCcardiomyocytes where myh6 expression associates with pacemaker- and atrial-like cells.30 For HUES-7, HUES-7TK/GCV and HUES-7R/puromycin the amplitude of ‘ventricular-like’ cardiomyocytes was 40.6 ± 6.2, 44.0 ± 7.4, and 39.3 ± 10.5 mV (P = 0.91; one-way ANOVA), while duration was 788 ± 158, 910 ± 244 and 579 ± 49 ms (P = 0.66; one-way ANOVA). For ‘non-ventricular-like’ cardiomyocytes the amplitude was 25.2 ± 7.3, 32.2 ± 3.9 and 22.8 ± 3.4 mV (P = 0.56; one-way ANOVA), while duration was 308 ± 29, 276 ± 77 and 308 ± 29 ms (P = 0.95; one-way ANOVA). Together these data demonstrate that drug treated cardiomyocytes retained functionality. Immunostaining of unselected HUES-7TK preparations showed that relative to total nuclei, 5.0 ± 1.4%, 38.3 ± 4.1% or 2032
1.1 ± 0.7% of cells stained with α-actinin, Ki67 or both markers, respectively. In contrast, the number of α-actinin positive cells increased significantly (P = 0.004; t-test) by 6.7-times to 33.4 ± 2.1% with GCV treatment (Figure 6d and e). Nevertheless, 66.6 ± 2.1% of the cells remaining in GCV selected HUES-7TK cultures were non-cardiomyocyte/non-proliferating and did not stain with α-actinin (Figure 6e). Puromycin treatment of HUES-7F preparations for up to 10 days failed in the enrichment of cardiomyocytes. Conversely, parallel treatment of HUES-7R preparations resulted in 14.5-fold enrichment with the percentage of α-actinin positive cells increasing significantly from 6.3 ± 3.4% in untreated cultures to 91.5 ± 4.3% in puromycin treated cultures (P < 0.001; t-test; Figure 6f and g). Specific enrichment of these cardiomyocytes was also confirmed by RT-PCR, where mesodermal (MYH6) but not endodermal (α-fetoprotein, AFP) or ectodermal (neural cell adhesion molecule 1, NCAM; neurofilament, NEFH) marker expression could be detected (Figure 6h). Therefore, expression of PAC from the MYH6 (αMHC) promoter is a feasible approach for transgenic enrichment of functional hESC-cardiomyocytes. www.moleculartherapy.org vol. 15 no. 11 nov. 2007
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Discussion Heterogeneity of differentiated populations derived from hESCs represents a considerable hurdle for the widespread use of these cells in scientific and clinical applications. This highlights the requirement for improved strategies to induce and direct differentiation, as well as to enrich for the desired lineage(s). Previously, we described standardized, feeder-free culture conditions that functioned between several hESC lines and developed a high throughput system that employed the forced aggregation of defined numbers of hESCs for forming EBs, which enabled the cardiomyogenic induction potential of growth factors to be tested.1,2 In this report we have shown that these technology platforms provide a relatively simple method for both transgenic modification and differentiation of hESCs that avoids the sequestration of transfection reagents by feeder cells and negates the need for drug resistant feeders.1 This method allowed for a 6.7- or 14.5-fold transgenic enrichment of hESC-cardiomyocytes via negative or positive selection strategies, respectively. To facilitate choosing an appropriate positive selection strategy, we examined expression of cardiac related genes in differentiating HUES-7 cells and observed a pattern similar to that reported in HES-213 and H9.2,12 as well as that for mouse embryonic development.31 The MYH6 gene showed robust expression in HUES-7 EBs and was upregulated by 65-fold from day 8 to 16 of differentiation. Increases in MYH6 transcripts have also been documented during differentiation of Miz-HES2 or HSF6, although upregulation was 80% of cells, comparable with that described for H1, 7, 9, and 14 cultured on Matrigel, where the percentage of SSEA-4 positive cells typically ranged from 73 to 99%.40 The EB size on day 4 of differentiation was also highly reproducible, underscoring the fact that a forced aggregation of defined numbers of cells can not only be used to improve differentiation but also to make quantitative interline comparisons.2 Analysis of FP recordings of beating EBs showed that basal beat rates were similar between the lines and did not appear to be influenced by the stage of differentiation (data not shown). Interestingly, studies in cardiomyocytes from H7 have demonstrated an increase in beat rate from day 10 to 45 of differentiation11 but others have reported threefold decreases for H9 and H14 from day 30 to 907 or ninefold for H1 from day 10 to 60,6 possibly reflecting intrinsic differences between hESC lines, differentiation strategy or culture practice. Incubation of HUES-7 beating areas with increasing doses of isoprenaline induced an increase in the beat rate, corroborating reports from a range of other hESC lines.3,5–7,11 This response confirms the presence of cardiomyocytes since isoprenaline has no effect on skeletal myotubes and inhibits contraction of smooth muscle.11 However, isoprenaline treatment of HUES-7 beating EBs did not elicit any significant increase in the amplitude (FPmax or FPmin), which contrasts with the response of H1 derived cardiomyocytes where 1,000 nmol/l isoprenaline invoked a 1.7-fold increase in FPmin. These differences in FP morphology may reflect differences in the proportions of ventricular-, atrial- or nodal-like cardiomyocytes derived from HUES-7 versus H1. Indeed, previous studies assessing electrophysiology by patch-clamp analysis demonstrated that while 85% of HES-2 derived cardiomyocytes displayed a ventricular phenotype,3 the figure was 52% from H9 or H147 and clearly this issue will require detailed investigation in future studies. 2033
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Constitutive expression of GFP and PAC from the pCAG promoter in HUES-7GIP cells or of HSVtk and/or neomycin phosphotransferase from PGK promoters in HUES-7TK, HUES-7F, or HUES-7R was maintained both in undifferentiated and differentiating cells. This demonstrates that these promoters can sustain long-term expression in hESCs and their differentiated derivatives, and that these transgenes are not detrimental to cell viability. Notably, the ability of GCV to ablate proliferating cells from dissociated HUES-7TK beating areas led to a >6-fold enrichment of cardiomyocytes and this should facilitate longterm in vitro studies without the interference from culture overgrowth.6,12 Nevertheless, in our studies GCV treatment on ~day 25 of differentiation also eliminated an important but minor population of proliferating cardiomyocytes. In future this could be countered by delaying GCV treatment to a later point of time thus maximizing the numbers of cardiomyocytes produced whilst retaining the ability to ablate the unwanted, proliferating non-cardiomyocyte populations; this is because the percentage of hESC-cardiomyocytes engaged in the cell cycle is reported to rapidly decrease during differentiation (~55% Ki67+ on day 20 decreasing to close to 0% by day 36 in line H9.29). Interestingly, puromycin treatment of disaggregated HUES-7F beating areas led to the death of >98% cells but no enrichment of the cardiomyocyte fraction was observed. This is consistent with the failure to detect expression of the GFP-IRES-PAC transgene by RT-PCR in beating areas derived from HUES-7F. While the precise reason for transgene inactivity is not known, the most probable cause is interference of the MYH6 promoter function by the PGK-Neo-pA cassette, as reported for other transgenic systems.41 This underscores the importance of testing expression cassettes in both orientations before drawing final conclusions on the effectiveness of a transgenic strategy. In contrast, in dissociated HUES-7R beating areas, where the orientation of the PGK-Neo-pA cassette was reversed, puromycin treatment resulted in a 14.5-fold enrichment to produce cultures in which >90% of the cells were cardiomyocytes. Surprisingly, GFP fluorescence was not detectable in these cells, implying that the strength of the MYH6 promoter is insufficient to produce visible levels of GFP but is sufficient to support puromycin resistance. It is reasonable to assume that this discrepancy arises because a single molecule of the PAC enzyme can process multiple puromycin molecules and so provides an additional level of functional amplification not available to GFP. Indeed, we have also observed considerable variation in GFP fluorescence (from undetectable to high levels) in puromycin resistant mouse ESC clones transfected with pCAG-GFP-IRES-PAC (C.D., unpublished results). Although GFP is not essential in the current strategy, the use of hybrid promoters (e.g., cytomegalovirus enhancer/MLC2v promoter) in cardiomyocytes derived from mouse embryonic stem or embryonal carcinoma cells has been shown to increase GFP expression tenfold from undetectable to detectable levels25,26 and may be relevant for future studies on hESCs. In conclusion, we have provided first proof of principle that transgenic enrichment of hESC-cardiomyocytes by negative or positive selection strategies is feasible. Both systems can be readily employed and will be of generic utility for enrichment of many differentiated lineages. The HSVtk/GCV system mediates toxicity 2034
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only in actively dividing cells and is therefore ideally suited for enriching terminally differentiated, non-dividing hESC-derived cell types such as cardiac and neuronal lineages. The use of lineagespecific promoter/PAC fusion constructs will be applicable to all hESC-derived cell types, provided suitable promoters are available. The next challenge will be to determine the most effective route to scaling up production of hESC-cardiomyocytes to test functional integration in animal models with cardiac dysfunction.
Materials and Methods All tissue culture reagents were obtained from Invitrogen (Paisley, UK; http://www.invitrogen.com/) and chemicals were from Sigma (Poole, UK; http://www.sigmaaldrich.com/), unless otherwise stated. The medium was changed daily for hESC culture and every 3–4 days during differentiation. HUES-7 culture and transfection. The hESC line HUES-720 was cultured by
trypsin-passaging in feeder-free conditions on Matrigel in mouse embryo fibroblast conditioned medium as described previously.2 To optimize transfection, 1 × 105 HUES-7 cells were seeded on Matrigel-coated 12-well plates and allowed to attach overnight. Different lipid transfection reagents were incubated at a ratio of 3:1, 2:1 or 1:1 with pGIP (a pCAG-GFP-IRES-PAC-pA plasmid that has a GFP-IRES-PAC cassette under the control of chicken β-actin/cytomegalovirus hybrid promoter21), using the protocols supplied by each manufacturer. The lipid transfection reagents used were GeneJammer (Stratagene, Amsterdam, Netherlands; http://www.stratagene.com), Metafectene (Biontex, Planegg, Germany; http://www.Biontex.com), Fugene (Roche, Burgess Hill, UK; http://www.roche-applied-science.com), ExGen 500 (Fermentas, York, UK; http://www.fermentas.com), GeneJuice (Novagen, Nottingham, UK; http://www.merckbiosciences.co.uk), Lipofectamine 2000 (Invitrogen). Electroporation was also performed with 1 × 106 cells using previously optimized setting of 600 V/30 µs (Multiporator; Eppendorf, Cambridge, UK; http://www.eppendorf.com). Forty-eight hours after transfection, 20,000 cells/sample were trypsinized and analyzed by fluorescenceactivated cell sorting (CoulterAltra flow cytometer; Becton Dickinson, Oxford, UK; http://www.bdeurope.com). Plasmid pTK has two expression cassettes. PGK promoter-NeoR-PGK
pA and PGK promoter-HSVtk-PGK pA.22 Plasmids pMYHF and pMYHR were constructed by cloning a 2.4 kilobase (kb) AgeI-PstI GFP-IRES-PACpA fragment from pGIP to modified pBluescript (Stratagene). A 4.5 kb BglII fragment containing the human MYH6 (αMHC) promoter was excised from pGL3-MYH6-I23 and cloned into a BamHI site upstream of GFP. Finally, a 1.5 kb XhoI PGK promoter-NeoR-PGK pA fragment from pTK was cloned into a unique SalI site in forward and reverse orientations to generate pMYHF and pMYHR, respectively. pGIP, pTK, pMYHF and pMYHR were all linearized with AhdI for stable transfection. The HUES-7TK line was generated by transfection with GeneJammer.
pTK at a ratio of 3:1. Electroporation (600 V/30 µs) of 20 µg of pGIP, pMYH6F or pMYH6R was used to generate HUES-7GIP, HUES-7F or HUES-7R. Forty-eight hours after transfection, selection with 150 µg/ml G418 (HUES-7TK, HUES-7F and HUES-7R) or 300 ng/ml puromycin (HUES-7GIP) was initiated and maintained continuously thereafter, including during differentiation. Transgenic HUES-7 lines were established by pooling drug resistant colonies and G-banding analysis2 of 60 chromosome spreads per line indicated karyotypic normalcy (46,XY; data not shown). PCR analysis. Transgene integration and expression, as well as expression
of endogenous genes in undifferentiated HUES-7 cells, EBs and cardiomyocytes was determined by PCR or RT-PCR. Genomic DNA was isolated by overnight lysis (50 mmol/l Tris, pH 8, 20 mmol/l EDTA, 100 mmol/l NaCl, 0.3% sodium dodecyl sulfate, 10 µg/ml proteinase K), followed by www.moleculartherapy.org vol. 15 no. 11 nov. 2007
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phenol/chloroform extraction and ethanol precipitation. Pellets were resuspended TE (10 mmol/l Tris–HCl, 1 mmol/l EDTA, pH 8). For qualitative RT-PCR, reverse transcription was carried out using Superscript II (Invitrogen) with 400 ng RNA, according to manufacturers’ instructions. Primers and cycle conditions are in Supplementary Table S2. Taqman quantitative PCR was carried out using Applied Biosystems Assay on Demand primers/probe sets to MYH6 (Part number Hs00411908_m1) and HPRT (internal control, Hs99999909_m1) in conjunction with TaqMan Universal PCR Master Mix, No AmpErase UNG (Applied Biosystems, Warrington, UK; http://www.appliedbiosystems.com/) and 2 µl complementary DNA in a total reaction volume of 20 µl. Cycle conditions were 95 °C for 5 minutes, 1 cycle followed by 95 °C for 10 seconds/60 °C for 1 minute, 50 cycles. Two independent PCR reactions, each in triplicate, were run and relative quantification performed using Applied Biosystems 7500 Fast Real-time PCR System & software. Cardiomyocyte differentiation and selection. EB formation and culture
was performed by forced aggregation by centrifugation of 3,000 HUES7 cells per well of untreated V-96 plates as described previously.2 Beating EBs were identified from day 11 of differentiation onwards and used for DNA or RNA extraction, cardiomyocyte selection and electrophysiological analysis. Sensitivity of undifferentiated HUES-7 or HUES-7TK to GCV was evaluated by seeding 1 × 104/cm2 cells in conditioned medium containing 0, 0.01, 0.1, 1, 10, or 100 µmol/l GCV. Medium changes were carried out daily using conditioned medium with the appropriate concentration of GCV. Cells were harvested for counting on day 5 of treatment. The same concentration range and treatment period was used on differentiated HUES-7 or HUES-7TK cultured in Dulbecco’s modified Eagle’s medium supplemented with 20% fetal bovine serum, 1% non-essential amino acids, 2 mm glutamine and 100 µm β-mercaptoethanol; cells were derived by disaggregating EBs on day 25 of differentiation using a three step protocol, as described previously.2,24 The ability of GCV or puromycin to enrich cardiomyocytes from HUES-7TK or HUES-7F/R, respectively, was tested. Cells from disag gregated beating HUES-7TK EBs were incubated with or without 10 µmol/l GCV for 7 days. Alternatively, cells from disaggregated beating HUES-7F or HUES-7R EBs either were left untreated or were treated with 100–200 ng/ml puromycin for 3, 5, or 7 days. After treatment all samples were fixed in 4% paraformaldehyde for immunostaining. FP analysis by micro electrode array. For electrophysiological extracel-
lular FP measurements, whole beating EBs were plated directly onto Matrigel-coated Micro Electrode Arrays (Scientifica, Uckfield, UK; http:// www.scientifica.uk.com/). Each micro electrode array culture dish contains 60 titanium nitride electrodes, with one of them acting as a reference electrode. Electrodes are 30 µm in diameter and spaced at 200 µm intervals. Contact pads and tracks are made of indium tin oxide, which is light transmissible thereby allowing visualization of the beating areas. All recordings were made at 37 °C in Dulbecco’s modified Eagle’s medium supplemented with 20% fetal bovine serum, 1% non-essential amino acids, 2 mm glutamine and 100 µm β-mercaptoethanol using 10 kHz sampling rate and captured using the Multi Channel Systems data acquisition system (Reutlingen, Germany; http://www.multichannelsystems.com/). Basal measurements as well as measurements made for escalating doses of isoprenaline (10−9 mol/l to 10−6 mol/l; Tocris, Bristol, UK; http://www.tocris. com) in the absence or presence of propranolol (10−7 mol/l; Sigma) were made. Data were analyzed off-line to determine interspike interval, FPmax (peak-to-peak amplitude) and FPmin (minimum potential). Whole-cell patch-clamp analysis. Whole-cell patch-clamp recordings
were obtained using an Axopatch 200A patch-clamp amplifier (Axon Instruments/Molecular Devices, CA) in normal current-clamp mode. Cardiomyocytes cultured in 35 mm MatTek dishes (with glass coverslip Molecular Therapy vol. 15 no. 11 nov. 2007
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insert; MatTek, Ashland, MA; http://www.mattek.com) were transferred to the stage of an inverted microscope and bathed in Dulbecco’s modified Eagle’s medium supplemented with 20% fetal bovine serum, 1% nonessential amino acids, 2 mm glutamine and 100 µm β-mercaptoethanol maintained at 34 °C using a heated stage (Model HCMIS, ALA, New York) and controller (Model PTC-10, npi Electronic, Tamm, Germany). Pipettes were pulled on a Sutter P-97 programmable micropipette puller and had resistances of 5–10 MΩ when filled with solution containing (mmol/l) 145 KCl, 5 NaCl, 2 CaCl2, 2 MgCl2, 4 ethylene glycol tetraacetic acid, 10 HEPES, pH 7.3. Data were transferred to a PC via an A/D interface (Model PCI6014, National Instruments) and recorded and analyzed using WinEDR v2.5.7 software (Dr. John Dempster, Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, UK). Immunostaining. For assessment of pluripotency, samples were fixed with
4% paraformaldehyde and permeabilized with 0.1% Triton-X100, then incubated with OCT4 antibody (1:200; Santa Cruz Biotech, Heidelberg, Germany; http://www.scbt.com/) for 1 hour at room temperature. Cy3conjugated goat anti-mouse IgG + IgM secondary antibody (1:250; Jackson Immuno Research, West Grove, PA; http://jacksonimmuno.com/) was applied for 45 minutes at room temperature. Alternatively, live cells were incubated with SSEA-4 antibody (1:50; Chemicon, Chandlers Ford, UK; http://www.chemicon.com/) for 15 minutes at 37 °C followed by Cy5conjugated goat anti-mouse IgG + IgM secondary antibody (1:200; Jackson Immuno Research) for 15 minutes at 37 °C and then 50,000 cells/sample were analyzed by fluorescence-activated cell sorting (CoulterAltra flow cytometer, Becton Dickinson). Cells from dissociated beating EBs were incubated with mouse monoclonal anti-α-actinin (1:800; Sigma) with or without counterstaining with rabbit polyclonal anti-Ki67 (1:100; Abcam, Cambridge, UK; http://www.abcam.com/) for 1 hour at room temperature. Incubation with the secondary antibodies, Cy3 goat anti-mouse IgG + IgM (1:250) and fluorescein isothiocyanate Goat-anti-Rabbit IgG (1:1,000; Vector Labs), was for 1 hour at room temperature. Samples were mounted in Vectorshield Hardset containing 4′,6-diamidino-2-phenylindole (Vector Labs, UK; http://www.vectorlabs.com/) and visualized on a Leica SP2 confocal microscope.
Acknowledgments The Medical Research Council, Biotechnology and Biological Sciences Research Council, and the University of Nottingham funded this study. The authors would like to thank Lorraine Young (University of Nottingham) for her valuable discussions on the manuscript.
Supplementary Material Figure S1. Optimization of HUES-7 cell transfection. Table S1. Analysis of beat rates from multi electrode array data. Table S2. Primers for PCR analysis.
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