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Additional Supporting Information may be found in the online version of this article.

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CYTO_22090_sm_SuppFig1.tif6296KSupplementary Figure 1. Pluripotency marker expression in undifferentiated pluripotent stem cells. (A) Collagenase treated hESC (H9; passage 50) and fibroblast-hiPSC (IMR90-4; passage 66) were passaged routinely onto MEF, prior to hEB differentiation (1st row: day 1, 2nd row: day 3, 3rd and 4th rows: day 6 after passage). Pluripotent stem cell lines generated colonies with well-circumscribed edges and blast cells with high nuclear to cytoplasmic ratios. The scale bars represent 100 μm. (B) hESC and hiPSC colonies were enzymatically digested to single cell suspensions and stained with antibodies to SSEA4, TRA-1-60, TRA-1-81, OCT-4, and NANOG. IMR90-4 cell line showed similar or higher expression of all pluripotency markers compared with H9. Shown are representative experiments performed twice.
CYTO_22090_sm_SuppFig2.tif1089KSupplementary Figure 2. Validation of the viability gating strategy. Samples were analyzed using a dual-laser FACSCaliburTM flow cytometer. Shown is a representative analysis of adherent EGM-2 cultured hEB derived cells. Live gating was approximated in all our set of experiments using light scatter properties (linear SSC x linear FSC, left histogram, region A). Cell viability was assessed using the LIVE/DEAD® Fixable Red Dead Cell stain (Invitrogen). Less than 1% of residual dead cells were still detected after light scatter-based viability gating (Gate A, center). By comparison, approximately 5% of all ungated events were detected as dead cells (positive in the FL3 channel, right), matching pre-staining cellular viability of 90 to 95%. Data files were analyzed using Flowjo analycal software.
CYTO_22090_sm_SuppFig3.tif7709KSupplementary Figure 3. Hematopoietic cells arise from the endothelial layer of EGM-2 culture. Day 8 hEB with addition day 3 in EGM-2 culture generate clumps of cells (A) generating endothelial layers with immature (B, cobblestone morphology) and mature (C, granulated shapes and soon to be release to medium) hematopoietic colonies.
CYTO_22090_sm_SuppFig4.tif2729KSupplementary Figure 4. Clonogenic hematopoietic cobblestones arose from endothelial layer include cells expressing RUNX1 (red), the transcription factor that is critical for definitive hematopoiesis. RUNX1+ cells were prominent but rare. Scale bars are 50 μm.
CYTO_22090_sm_SuppTab1.tif580KSupporting Information Table 1. The medium composition for culture of hESC/hiPSC and MEF.
CYTO_22090_sm_SuppTab2.tif612KSupporting Information Table 2. The medium composition for generating hEB and differentiation into hemato-vascular lineages.
MIFlowCyt-Item-Location.doc48KSupporting Information: MIFlowCyt

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