Effects of iron deficiency on in vitro TPO induced megakaryopoiesis. To evaluate the effects of iron deficiency on TPO-induced megakaryopoiesis (in the absence of ELT), CD34⁺ cells were cultured for 14 days in medium supplemented with either 100% HOLO, 100% APO, or no transferrin and 50 ng/mL of TPO to stimulate maximal proliferation and maturation. (A) Bars display the percentage of cells in every condition and timepoint related to the number of CD41⁺ cells generated at the end of 14 days in cultures with 100% HOLO (set at 100%). Cell expansion was reduced in cultures containing 0% HOLO compared with those with 100% HOLO, starting on day 11 of culture. (B) The percentage of CD41⁺ cells was also reduced by iron deficiency, with severe reductions in cultures without transferrin. (C) The maturation of committed MKs, indicated as the percentage of CD42b⁺ cells in the CD41 positive population, was not influenced by iron status. (D) Shown are representative photomicrographs of cells on day 14 of culture. Cells in 100% APO were reduced in number, and cultures without transferrin exhibited extensive cell death. Images present the average of 4 independent experiments. *P < .05; **P < .01; ***P < .001.