Bitopertin increases the erythroid expansion of marrow from patients with DBA in culture. (A) Blue fluorescent protein positive sorted CD34⁺ cells expressing either control (shLuc) or RPS19 short hairpin RNA (shRNA) were cultured for 10 days in differentiation media I without or with 10 nM bitopertin (left). The erythroid precursors were then cultured in an EBI culture with CD14-derived macrophage for an additional 3 days in differentiation media II without or with bitopertin (right). Data are presented as the mean and standard error of the mean of the numbers of cells recovered from 3 independent experiments (control: n = 3, RPS19 shRNA: n = 6 [3 each of 2 independent shRNAs]) after normalizing cell counts to reflect starting each culture with 200 000 cells. (B) Characteristics of marrow samples from patients with DBA and control subjects used in studies. (C) Representative flow cytometry analyses of cryopreserved marrow mononuclear cells from a patient with DBA (D2) and a control subject (N1) before and after 3 days in erythroid culture show that erythroid differentiation in patients with DBA fails in vivo at the colony-forming unit erythrocyte and/or proerythroblast stage when CD71 is upregulated, but in vitro culture allows continued differentiation. (D) A representative time course showing the total cell expansion and CD71⁺ cell expansion of marrow cells from normal (N1) and a patient with DBA (D1) cultured without or with bitopertin. (E) Total cell expansion or expansion of CD71⁺ cells in marrow cultures from control subjects and patients with DBA for 10 days in differentiation media I without or with bitopertin. Cell counts from each sample were normalized to the culture without bitopertin and presented as a percent of control (n = 4). ∗P < .05; ∗∗∗P < .001; ∗∗∗∗P < .0001; ns, P > .05. ns, not significant.