Table 1. Culture protocol.

Phase	Culture period	Feeder cells	Attached cells	Detached cells	Specific factors used
I	Day 0	OP9 cells	(Start)	(Start)	VEGF, IGF-II
II	Day 4	Change to new cells	Discarded a	Re-cultured b	SCF, EPO, IL-3, Dex
II	Day 7	No change	Remained c	Re-cultured d	SCF, EPO, IL-3, Dex
II	Day 10∼e	Change or No change f	Remained or Discarded g	Re-cultured h	SCF, EPO, IL-3, Dex
III	Day 60∼i	(-) j	(-) k	Re-cultured h	SCF, EPO, IL-3, Dex
IV	Day 120∼l	(-) j	(-) k	Re-cultured h	Essential factors l

To induce hematopoiesis, 5×10⁵ ES cells were cultured on feeder cells with cytokines in two 100 mm-dishes, with 2.5×10⁵ ES cells per dish. Phase I∼IV, four different phases of culture. Attached cells and Detached cells, the cells derived from ES cells and attached to feeder cells or detached from feeder cells. VEGF, vascular endothelial growth factor. IGF-II, insulin-like growth factor-II. SCF, stem cell factor. EPO, erythropoietin. IL-3, interleukin-3. Dex, dexamethasone.

a, the attached cells were discarded together with the used feeder cells. b, the detached cells collected from two dishes were cultured again on new OP9 cells in a 100 mm-dish. c, the attached cells were cultured further without any treatment. d, all detached cells collected from a dish were cultured again. e, medium changes were performed twice a week. f, when the attached cells reached approximately 80% confluence, feeder cells were changed to new OP9 cells. g, when the feeder cells were changed to new cells, the attached cells were discarded together with the used feeder cells. h, all detached cells collected from a dish were cultured again, or a portion of detached cells were cultured again and other detached cells were subjected to analyses or discarded. i, approximately as of Day 60 we started to try the culture in the absence of feeder cells using a portion of the detached cells, simultaneously continuing the culture in the presence of feeder cells as the Phase II culture. j, no feeder cells were used in the Phase III and IV culture. k, the cells attached to the dish were barely detected. l, approximately as of Day 120 the essential factor(s) for proliferation was evaluated, and then each cell line was cultured in the presence of the essential factor(s) alone.