hES cells-derived mesoderm progenitors further differentiate in vitro. (A) hES cells were differentiated with BMP-4 (B + Noggin) or without BMP-4 (Null + Noggin) for 24 hours; then cells were washed and transferred to SFM containing Noggin. Three days after that, SFM was replaced by SCM without Noggin. Then cells were collected at indicated days and analyzed for cardiac gene expression by RT-PCR. (B) hES cells were differentiated with BMP-4 (B) or without BMP-4 (Null) for 24 hours; then cells were washed and transferred to SFM supplemented with bFGF (F) or without bFGF (Null). Three days after that, SFM was replaced by SCM without bFGF. The cultures were then analyzed for hematopoietic differentiation by flow cytometry analysis of KDR and CD34 at days 4 and 6, and days 8 and 10, respectively. Numbers indicate percentage of KDR⁺ or CD34⁺ cells in each culture. (C,D) Statistical analysis of KDR⁺ cells at days 4 and 6 (C) and CD34⁺ cells at days 8 and 10 (D) under different differentiation conditions. Values are mean and SD from 3 independent experiments (**P < .01, ***P < .001). (E) Hematopoietic progenitor capacity of CD34⁺ and CD34⁻ subsets from differentiated hES cells with or without BMP-4 treatment. Values are mean and SD from 3 independent experiments. (F) Examples of different CFU colonies, including (i) CFU-erythroid (CFU-E), (ii) burst-forming unit-erythroid (BFU-E), (iii) CFU-myeloid (CFU-M), (iv) CFU-granulocyte, macrophage (CFU-GM), and (v) CFU-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM). (G-H) hES cells were differentiated in the presence of 25 ng/mL BMP-4 for 7 days (B25), then analyzed for cardiac gene (G) and hematopoietic gene (H) expression. And hES cells differentiated for 7 days as described in panels A and B (B + Noggin, B + F) were used as positive controls for cardiac and hematopoietic gene expression. Control indicates isotype control. Scale bars represent 50 μm (Fi), 100 μm (Fii,iii,v), and 200 μm (Fiv).