Optical mapping of membrane potentials and immunostaining in CPC-NRVM cocultures Electrical propagation in CPC/NRVM cocultures was optically mapped using a bundle of 504 hexagonally arranged optical fibers (RedShirtImaging) providing a spatial resolution of 750 mm in a 19.5 mm field of view, as previously described [18,50]. Briefly, the cocultures were incubated for five minutes with a voltage sensitive dye (di-4 ANEPPS, 16 mM) at room temperature, placed in a heated recording chamber, perfused with Tyrode’s solution, and illuminated with a green excitation light (520630 nm). The recorded red fluorescence (.590 nm) was transferred through the fiber optic bundle, converted to voltage using photodiodes, amplified, sampled at 2.4 kHz, and stored on a PC. Before the onset of pacing, all cocultures were checked for the presence of spontaneous activity. A XYZ-micropositioned bipolar platinum electrode was then used to locally stimulate the CPC area in the center of the monolayer and propagated action potentials were optically mapped. Electrical stimulation, light exposure, and data acquisition were synchronized using custom LabView software. Data analysis was performed using MATLAB as previously described [18,51]. Activation times were defined as times of 50% upstroke of the action potential and used to construct isochrone maps. Local conduction velocities were calculated using the activation time for each optical channel relative to those of neighboring channels[18]. Following optical mapping analysis the samples were fixed in 4% parafolmaldehyde and stained with antibodies against the GFP protein (Molecular probes, A21311, A11120), a-Actinin (Sigma, A7811), Acta2 (Abcam, ab5694), and Connexin 43 (Sigma, C6219). Secondary antibodies were purchased from Molecular probes and were conjugates to the fluorochromes Alexa 488, and Alexa 546. Nuclei were stained with 49,6-diamidino-2-phenylin- dole (Molecular probes). The samples were imaged with a Nikon TE2000 and a confocal Zeiss 510 microscope. Animal Model All research involving experimentation on vertebrate animals have been specifically approved by Johns Hopkins Medical Institutions (Animal Protocol # MO0M296). Using a left anterior thoracotomy, the heart of SVE129 female mice (8–10 weeks of age) was exposed and LAD was ligated permanently. For sham surgeries, the needle was passed under the LAD without ligation. The mice were monitored twice a day for four weeks. Four experimental groups were established consisting of animals that underwent LAD ligation and received phosphate buffered saline injections with (n = 15) or without cells (n = 10) and animals that underwent sham surgeries and also received saline injections with (n = 7) or without cells (n = 11). Immediately following infarction injury, each animal received three injections (10 ml/injection) of saline or saline with CPCs (0.5–16106 cells total) in the boundaries and the center of the infarcted region. The cells used in these experiments were GFP+ CPCs isolated on differentiation day 6. The animals were sacrificed four weeks post-transplantation and the hearts prepared for immunohistological analysis. CPC injections Using a tubing system attached to a standing syringe, we injected 4–56105 cells in three doses (10 ml/injection) directly into myocardium with a 30-gauge needle. Injections were done immediately following LAD ligation, into the mid infarct zone and border-zones. The infarct zone was defined by blanching of the heart after ligation. Progenitor Cell Heart Therapy Echocardiography The analysis was done using a HP/PHILIPS/AGILENT SONOS 5500. The animals were analyzed prior to surgery (same day) and at 1, 2, and 4 weeks after it. The procedures were performed under general anesthesia using isoflurane 1–2%. Pressure volume loop analysis 4 weeks after the surgery the mice were anesthetized using a combination of etomidate, morphine and urethane, which were injected intraperitoneally. Using the right carotid approach a Millar SPS 839 catheter progressed to the left ventricle. During the procedure, a diluted 6.25% albumin solution was infused into jugular vein at a rate of 5 mcl/min. Pressure volume data were obtained at baseline with closed chest and after opening the thorax with IVC occlusion. The volume calibration was done using thoracic aortic flow and hypertonic saline infusion. Statistical analysis We obtained average and standard error of mean for all values and used one way and two way ANOVAs for statistical analysis (Graph-pad prism 4.03). Supporting Information File S1 Cell culture and differentiation, FACS analysis and sorting, isolation of nrvms, harvesting the hearts and tissue preparation, immunocytochemistry, statistical analysis. Found at: doi:10.1371/journal.pone.0011536.s001 (0.06 MB DOC) Figure S1 Cardiomyocyte and smooth muscle lineage commit- ted mouse ESC-derived CPCs functionally electrocouple with neonatal rat ventricular cardiomyocytes. (a–b) In vitro co-culture of ESC-derived CPCs with NRVMs. The CPCs were selectively plated at low density in the center region of the coverslip and subsequently differentiated into cardiomyocytes and smooth muscle cells. A confluent layer of NRVMs surrounded the CPCs. Dashed lines denote the border between the two cell types. Mouse ESC-derived cardiomyocytes as well as NRVMs stain positive for a-Actinin (red), whereas only mouse ESC-derived CPCs and mouse ESC-derived cardiomyocytes express GFP (green). (c–d) High magnification immunostaining analysis illustrates the interface of the two cell types demonstrating formation of Connexin 43 gap junctions. (e) The majority of CPCs plated at low density differentiated into smooth muscle cells (e, green) with some cardiomyocyte differentiation (e, red). (f) Sequential isopotential maps reveal electrical propagation from the central island of differentiated CPCs to the outside regions containing primary cardiomyocytes (central island is indicated by dashed white circle). Color bar shows relative transmembrane potential normalized from resting (blue) to peak (red) value. The monolayer was electrically stimulated by a point electrode in the center, indicated by the yellow pulse sign in 0 ms frame. The timing index has been set relative to the stimulus pulse (indicated by the yellow triangle on membrane potential traces). Membrane potential traces are from three channels (channel 1 in the central island of cardiac progenitor cells and channels 2 and 3 in the outside region with cardiac cells). Note that electrical propagation was very slow inside the central island due to predominance of CPC-derived smooth muscle cells and significantly accelerated after exiting to the outside cardiomyocyte region. Found at: doi:10.1371/journal.pone.0011536.s002 (3.21 MB TIF) Figure S2 Kaplan-Meyer survival graph showing the four study arms during the four weeks after myocardial infarction. Logrank PLoS ONE | www.plosone.org 12 July 2010 | Volume 5 | Issue 7 | e11536