Iron status influences early MK differentiation and expansion in response to ELT. CB CD34⁺ cells were cultured in conditions of iron depletion (0% HOLO), iron deficiency (10% HOLO), and iron repletion (100% HOLO) and with TPO plus different ELT concentrations. Cell number and differentiation were analyzed on culture-day 7 by flow cytometry. (A) Representative results of flow cytometric analysis of CD34 (y-axis, PE) and CD41 expression (x-axis, APC) in cells cultured with TPO3 plus ELT30 and various levels of iron availability, showing increased CD41 differentiation with increased iron availability. (B) Distribution of cell populations based on CD34 and CD41 surface expression on day 7 of culture with TPO3 plus escalating concentrations of ELT. Each line represents different levels of iron availability in culture. (C) Absolute cell numbers of each population were calculated based on cell numbers and percentages and are shown based on ELT concentration and iron status. Each data point reflects the mean ± SD from 6 independent experiments.