Types of colony-forming cells enriched in the CD34⁺ EB cell fraction. A3-1 ES cells were induced to differentiate in methylcellulose culture in the presence of 100 ng/mL SCF, and 20 ng/mL VEGF (A, B2), or 100 ng/mL SCF, 10 ng/mL IL-3, and 1 U/mL EPO (B1). EBs were obtained on day 7, stained with RAM34, and subjected to cell isolation by FACS. (A) One of the staining results of SCF + VEGF EB cells is shown: (1) the scatter pattern of the RAM34 stained sample, (2) staining pattern of isotype control, and (3) that of RAM34, in dot plots. Regions for sorting CD34⁺ (right) and CD34⁻ (left) cell populations are indicated in boxes (2 and 3). Number in each box indicates percentage of total EB cells. Similar scatter pattern and staining pattern were obtained from EBs developed with SCF + IL-3 + EPO. (B) Erythro-myeloid CFC in the CD34⁺ and CD34⁻ cell populations. Average numbers of CFU-M, CFU-Mmix, BFU-E, CFU-Emix, and CFU-E in 10⁵ presorted EB cells, 10⁵ sorted CD34⁺ EB cells, and 10⁵ sorted CD34⁻ EB cells were determined. The average colony numbers from CD34⁺ cells and CD34⁻ cells were divided by corresponding numbers from presorted EBs to obtain enrichment factors for individual CFCs. Such enrichment factors obtained from three (SCF + VEGF) to four (SCF + IL-3 + EPO) independent experiments were then averaged to obtain average enrichment factor. Hatched bar indicates results for CD34⁺ EB cells, and open bar is for CD34⁻ EB cells. Factor 1 means no enrichment. Vertical line indicates standard deviation.