Figure 1.
MN1-TEL enhances cytokine-dependent growth of myeloid progenitors. (A) In vitro hematopoietic differentiation of ES cells. MN1-TELKI/WT ES cells treated with Cre or not or WT ES cells were cultured in methylcellulose-based media (MC-1) with stem cell factor (SCF) to generate embryoid bodies (EBs). After 15 or 18 days, cells from dissociated EBs were plated into MC-2 in the presence of SCF, interleukin 3 (IL-3), and IL-6. Numbers of myeloid colonies were scored after 10 days of culture. Mean ± standard error (SE) is shown (n = 3). Experiments repeated with 3 different ES cell clones yielded identical results. (B) Colony-forming efficiency of MC-2 ES-derived hematopoietic progenitors in MC-3. Colony-forming cells (CFCs) were harvested from the MC-2 and replated into MC-3 with the same cytokines (replating assay). Numbers of myeloid colonies were scored after 10 days. Mean ± SE is shown (n = 3). Experiments repeated in triplicate using different ES clones yielded identical results. (C) Colonies in MC-3. Representative pictures captured on day 10 of culture are shown. Images were obtained as previously described.12 (D) GFP expression in ES-derived CFCs. GFP expression was analyzed by flow cytometry (FCM) after 10 days of culture in MC-2. Solid line indicates KI-ES–derived cells; broken line, control WT-ES–derived cells. (E) Cytokine-dependent growth of MN1-TEL+ myeloid progenitors. MN1-TEL/Mx1-Cre BM cells treated with polyinosinic-polycytidylic acid (pI-pC) or not were plated in MC. After the MC-3, cells were harvested and grown in liquid cultures. Numbers of viable cells are plotted. • indicates IL-3 + SCF; ○, IL-3; ▴, SCF; and ▵, no cytokine. Mean ± SE is shown (n = 3). Experiments repeated in triplicate using different cells yielded identical results. (F) May-Giemsa (M-G) staining of MN1-TEL+ cells in culture. Image obtained as previously described.12 (G) Fluorescence-activated cell sorter (FACS) analysis of Sca-1/c-Kit expression of an MN1-TEL+ cell line.