Files in this Data Supplement:

• Figure I - (Adobe Acrobat file) (937 KB). FACS assessment and purification of cardiomyocytes at Flk-d6 using TMRM fluorescent staining (see Methods). A. FACS analysis of EMG7 ES cell-derived cardiomyocytes for MHC-GFP and TMRM fluorescence. Approximately 50% of MHC-GFP+ cardiomyocyte population could be detected as TMRM-high population. B. Cardiomyocyte differentiation from EMG7 ES cells. Almost all of the purified TMRM-high population (7.9% of total cells) was MHC-GFP+ cardiomyocytes. C. Cardiomyocyte induction from 20D17 iPS cells (left) and D3 ES cells (right). Percentages of TMRM-high cardiomyocytes were indicated.
• Figure II - (Adobe Acrobat file) (526 KB). Purified cardiomyocytes with TMRM fluorescent staining. TMRM stained cells (see Methods) on OP9 cells at Flk-d6 were subjected to FACS. TMRM-high or low cells (see supplemental Figure I online) were re-plated to gelatin-coated dish and cultured. Cells were stained with anti-cTnT antibody (red) and DAPI (blue) after 2 days. More than 80% of TMRM-high cells were positive for cTnT (upper). On the contrary, a vast majority of TMRM-low cells were negative for cTnT (lower). Bars=500 µm.
• Figure III - (Adobe Acrobat file) (237 KB). Analysis of vascular structure formation from mouse iPS cells in 3-D culture. Induced vascular-like structures were immunostained with CD31 (green) and SMA (red), and subjected to confocal microscopy to acquire Z-stack images. Induced vasculatures formed CD31+ endothelial tubes with true lumen inside (A, XZ and YZ images). SMA+ mural cells attached to the endothelial tubes (B, arrowheads). Blue (Y axis), yellow (X axis) lines indicate sliced passions. Bar=25 µm
• Figure IV - (Adobe Acrobat file) (570 KB). Differentiation of blood cells from iPS cells. iPS cell-derived Flk1⁺ cells were cultured on OP9 stroma cells in differentiation medium for 3days. A, FACS analysis for CD45 at Flk-d3. Approximately 8.5% of total cells were CD45-positive blood cells. B, Immunostaining for CD45. CD45+ blood cell-like small cell clusters were observed.
• Figure V - (Adobe Acrobat file) (173 KB). An example of transgene mRNA expressions in long term culture of iPS cell-derived Flk1⁺ cells on OP9 cells (RT-PCR). Total (upper), transgene (middle), and endogenous (lower) mRNA expression in undifferentiated iPS cells and cells after 1 and 2 months of differentiation on OP9 cells. Transgene expression showed various patterns during differentiation. Occasionally, upregulation or re-expression of transgenes including c-myc was observed in 1 or 2 months of culture.
• Supplemental Methods - (Microsoft Word file) (279 KB).
• Movie I - (MOV file) (2.2 MB).Time-lapse video for in vitro vascular formation. iPS cell-derived Flk1⁺ cell aggregate was cultured in type I collagen gel. Time-lapse video recording was performed every 15 minutes for 4 days.
• Movie II - (MOV file) (790 KB). A self-beating colony from iPS cells. Flk1⁺ cells were cultured on OP9 cells for 5 days.