ACTH enhances enucleation of erythroblasts derived from human HPCs.

(A) The neutralization of melanocortins by addition of the neutralising antibodies (nAbs) for ACTH and α-MSH into culture media on the M0 day and the M3 day. The enucleation rate on the M5 day was measured. *, P < 0.01. n = 3; error bars, s.e.m. (B) Cell number between M0 and M4 day. ACTH39 has no significant effect on the proliferation of erythroblasts. n = 3; error bars, s.e.m. (C) The representative images of definitive erythroid differentiation induced by melanocortins on the M5 day. The glycophorin A (GPA)-positive cells without nuclei are increased in number by treatment with 10 nM ACTH39. Control, vehicle (0.1% BSA); ACTH39, 10 nM ACTH39; GPA, APC (red); nuclei, Hoechst33342 (blue). White arrows, enucleated cells; White arrowheads, enucleating cells; Green arrows, normal erythroblasts; Green arrowheads, excreted nuclei. Bar, 20 μm. The enucleation rates in definitive erythroid cells by treatments with (D) ACTH39, (E) ACTH24, and (F) α-MSH between the M5 and M7 days. All melanocortins showed effects on the induction of enucleation by erythroblasts. *, P < 0.01; **, P < 0.001; ***, P < 0.0001, each point versus control in the same day. n = 3; error bars, s.e.m.