Kinetic analysis of hematopoietic development in H1/OP9 coculture. (A) Gene expression analysis of hematopoiesis-inductive transcription factors (SCL, GATA1) and hematoendothelial markers by RT-PCR. Triangular white pointers indicate the first day when a surface expression of respective hematopoietic markers was detected by flow cytometry. Dotted vertical lines show the timeframe of CFC emergence. Human- and mouse-specific GAPDH primers were used for positive control of human (hGAPDH) and MEF/OP9 (mGAPDH) RNA, respectively. (B) Early CD235a⁺CD34^– cells in H1/OP9 coculture. Representative FACS analysis shows a burstlike CD235a expression and gradual emergence of CD34⁺CD235a^+/– cells during 3 to 5 days of H1/OP9 coculture. Values within dot plots indicate percentage of cells in respective quadrants; 20 000 FACS events are displayed. Bar graphs show CFC potential and relative expression of CD235a, GATA-1, and SCL mRNA in FACS-sorted CD235a^–CD34^–, CD235a⁺CD34^–, and CD34⁺ cells on day 4 of H1/OP9 coculture. CFCs and mRNA levels were determined by MethoCult GF+ assay and qRT-PCR, respectively. The relative expression of each GAPDH-normalized target gene was calculated in comparison with undifferentiated H1 cells using the 2^ΔΔCt method. Results are the mean ± SD from 3 independent experiments. (C) Expression of hematopoietic markers during H1/OP9 coculture was analyzed by FACS within gated CD34⁺ cells and represented as a percentage of CD34⁺ cells (left y-axis). Dashed trend line shows total CFC counts (right y-axis). Results are the means from 3 independent experiments. (D) Representative FACS analysis of CD43⁺ cells in H1/OP9 coculture. Values within dot plots indicate percentage of cells in respective quadrants; 20 000 (total H1/OP9 cells), 5000 (gated CD34⁺ cells, day 5), and 10 000 (gated CD34⁺ cells, days 7, 9) FACS events are displayed.