Mm line shown in the overlay image. (b) Pluripotency marker 4-IBP expression is not effected by mitochondrially targeted GFP. GFP localised to the mitochondria is co-expressed with pluripotency markers Oct-4 and SSEA4. Images are 150 mm wide. Co-expression of GFP and pluripotency markers was confirmed by flow cytometry. Histograms show GFP positive cells also express Oct-4 and SSEA-4. (c) GFP intensity is not lost during down regulation of cell surface pluripotency marker TG30. (d) KMEL2 cells have a normal karyotype. doi:10.1371/journal.pone.0052214.gTracking Mitochondria during hESC DifferentiationFigure 3. LDS-751 stains human embryonic stem cell mitochondria based on membrane potential. (a) LDS-751 is co-localised with GFP in the KMEL2 mitochondria reporter line and does not overlap with the nucleus. Fluorescence intensities for each fluorophore were measured along the 160 mm line shown in the overlay image and plotted as distance vs intensity. (b) Mitochondria specific staining is lost when treated with a mitochondrial membrane depolarising agent valinomycin. Line profile analysis demonstrates LDS-751 no longer localised to the mitochondria after blocking mitochondrial membrane potential. The line profile in the overlay image represents 140 mm. doi:10.1371/journal.pone.0052214.gTracking Mitochondria during hESC DifferentiationFigure 4. Mitochondrial localisation during neural lineage differentiation. Neural lineage specific differentiation showing KMEL2 positive for (a) Nestin and (c-e) b-III-tubulin. b-III-tubulin positive cells show expanded localisation of mitochondria SMER-28 custom synthesis through dendritic outgrowths (c and e). bIIIT, b-III-tubulin. Scale bars in (b) are 1000 mm. All other images are 150 mm wide. Enlarged images in 1317923 (e) are shown in the boxed regions of (c) and (d). doi:10.1371/journal.pone.0052214.g250 mM or above had detrimental effects on cell number and mitochondrial membrane potential as assessed by JC-1 staining(Figure S1). Neither AICAR nor metformin increased the percentage of MIXL1 positive cells above untreated controlsTracking Mitochondria during hESC 
DifferentiationFigure 5. Variable mitochondrial localisation during lineage specific differentiation. (a) Mitochondria in hESC are localised near the nucleus. (b) Mitochondria in AFP positive endoderm lineage cells. Mitochondria in AFP positive cells display a granular, dispersed localisation through the whole cell. (c and d) Mitochondria in MIXL1 positive cells (Mesendoderm) display a densely packed, perinuclear localisation based on MitoTracker far red (c) and LDS-751 (d) staining. AFP, alpha fetoprotein. Images (a-c) are 150 mm wide. Line profile in (d) represents 120 mm. doi:10.1371/journal.pone.0052214.g(Figure 1a). To determine if any biogenesis agents could increase MIXL1 positive cells during cardiogenic mesoderm induction, spin embryoid bodies were differentiated using APEL medium [34] and growth factors BMP4, Activin A, VEGF and SCF. Increasing concentrations of both SNAP and AICAR increased the percentage of MIXL1 positive cells 17.33611.72 (p,0.05) and 13.41613.4 (p.0.05) respectively above controls (Figure S2) as well as the relative level of MIXL1 expression within the cells (Figure 1c). In order to determine a positive impact of biogenesis agents on MIXL1 expression, embryoid bodies were formed in the presence of biogenesis agents diluted in DMSO with and without the key growth factors for differentiation, BMP4 and Activin A. As expected removal of either BMP4 or A.Mm line shown in the overlay image. (b) Pluripotency marker expression is not effected by mitochondrially targeted GFP. GFP localised to the mitochondria is co-expressed with pluripotency markers Oct-4 and SSEA4. Images are 150 mm wide. Co-expression of GFP and pluripotency markers was confirmed by flow cytometry. Histograms show GFP positive cells also express Oct-4 and SSEA-4. (c) GFP intensity is not lost during down regulation of cell surface pluripotency marker TG30. (d) KMEL2 cells have a normal karyotype. doi:10.1371/journal.pone.0052214.gTracking Mitochondria during hESC DifferentiationFigure 3. LDS-751 stains human embryonic stem cell mitochondria based on membrane potential. (a) LDS-751 is co-localised with 
GFP in the KMEL2 mitochondria reporter line and does not overlap with the nucleus. Fluorescence intensities for each fluorophore were measured along the 160 mm line shown in the overlay image and plotted as distance vs intensity. (b) Mitochondria specific staining is lost when treated with a mitochondrial membrane depolarising agent valinomycin. Line profile analysis demonstrates LDS-751 no longer localised to the mitochondria after blocking mitochondrial membrane potential. The line profile in the overlay image represents 140 mm. doi:10.1371/journal.pone.0052214.gTracking Mitochondria during hESC DifferentiationFigure 4. Mitochondrial localisation during neural lineage differentiation. Neural lineage specific differentiation showing KMEL2 positive for (a) Nestin and (c-e) b-III-tubulin. b-III-tubulin positive cells show expanded localisation of mitochondria through dendritic outgrowths (c and e). bIIIT, b-III-tubulin. Scale bars in (b) are 1000 mm. All other images are 150 mm wide. Enlarged images in 1317923 (e) are shown in the boxed regions of (c) and (d). doi:10.1371/journal.pone.0052214.g250 mM or above had detrimental effects on cell number and mitochondrial membrane potential as assessed by JC-1 staining(Figure S1). Neither AICAR nor metformin increased the percentage of MIXL1 positive cells above untreated controlsTracking Mitochondria during hESC DifferentiationFigure 5. Variable mitochondrial localisation during lineage specific differentiation. (a) Mitochondria in hESC are localised near the nucleus. (b) Mitochondria in AFP positive endoderm lineage cells. Mitochondria in AFP positive cells display a granular, dispersed localisation through the whole cell. (c and d) Mitochondria in MIXL1 positive cells (Mesendoderm) display a densely packed, perinuclear localisation based on MitoTracker far red (c) and LDS-751 (d) staining. AFP, alpha fetoprotein. Images (a-c) are 150 mm wide. Line profile in (d) represents 120 mm. doi:10.1371/journal.pone.0052214.g(Figure 1a). To determine if any biogenesis agents could increase MIXL1 positive cells during cardiogenic mesoderm induction, spin embryoid bodies were differentiated using APEL medium [34] and growth factors BMP4, Activin A, VEGF and SCF. Increasing concentrations of both SNAP and AICAR increased the percentage of MIXL1 positive cells 17.33611.72 (p,0.05) and 13.41613.4 (p.0.05) respectively above controls (Figure S2) as well as the relative level of MIXL1 expression within the cells (Figure 1c). In order to determine a positive impact of biogenesis agents on MIXL1 expression, embryoid bodies were formed in the presence of biogenesis agents diluted in DMSO with and without the key growth factors for differentiation, BMP4 and Activin A. As expected removal of either BMP4 or A.