OBJECTIVETo explore a stable technology for inducing ES cell to committed hematopoietic differentiation.

METHODSThe effects of various inductive factors on BLast Colony-Forming Cell (BL-CFC) were investigated. In vitro differentiative system for ES cells was employed in this study.

RESULTSA high linear correlation between the number of D3.5 EB-derived cells plated and the number of blast cell colonies was developed, r = 0.9931. With high frequency of blast colonies observed (1.08-1.2 colonies per 100 cells). 20%-30% D4T conditioned medium (D4T CM) showed the most significant growth potentials of blast colonies. D4T CM, EPO or KL alone had no blast colony growth promoting effect (P > 0.05). But VEGF alone had high significant blast colony growth promoting effect (P < 0.001). However, any two factors combination from above four factors exerted better growth promoting effect than VEGF alone (EPO + D4T CM, P < 0.05; KL + D4T CM, P < 0.01; VEGF + D4T CM, P < 0.001). There were no significant difference among VEGF + KL and EPO + D4T CM or KL + D4T CM, and KL + D4T CM (P > 0.05). While the combination of VEGF + D4T CM was better than KL + D4T CM, VEGF + KL or EPO + D4T CM (P < 0.001). Moreover, the combination of VEGF + KL + D4T CM + EPO, had the highest significant blast colony growth promoting effect (P < 0.001). And the highest frequency of blast colonies was observed (1.5-1.2 colonies per 100 cells).

CONCLUSIONVEGF may be the main factor which stimulates the growth of significant numbers of blast cell colonies. D4T CM maybe contains strong cofactors. EPO and KL are the main factors for the induction of BL-CFC to committed hematopoietic differentiation. D3.5 EB-derived cells are more sensitive to various stimulators and have strong blast colony growth promoting effect than that of D3.25 EB-derived cells.