Figure 1
Figure 1. Formation of hematopoietic progenitors from day 14 EBs derived from control and Scl ESCs. (A) Control and Scl ESCs cultured in methylcellulose medium supplemented with hematopoietic cytokines (stem cell factor, interleukin-1, interleukin-3, granulocyte-macrophage colony-stimulating factor, erythropoietin) in the absence of leukemia inhibiting factor. Scale bar represents 150 μm. (B) Immunoblot analysis of total protein extracts prepared from day 14 EBs derived from wild-type and Scl ESC clones. (Top panel) Immunoblotting with probe targeting hemoglobin-α antigen (1:1000). (Bottom panel) Immunoblotting with probe targeting β-actin antigen (1:10 000). Lanes 2 and 6 (TD0) were spliced from another blot because of the extremely low yield of EBs from TD0 ESCs. The results are consistent from separate experiments. (C) Representative histograms of flow cytometric analyses on 2 clones from wild-type ESCs and 2 clones from Scl ESCs. CD45 marker was analyzed by the FL4 channel (y-axis), and CD11b marker was analyzed by the FL2 channel (x-axis). (D) Calculation of the CD45CD11b double-positive cell populations from each clone. All cell preparations were done in duplicates, and each cell population was averaged from 2 separate experiments. Error bars represent 1 SD. (E) CD11b immunostaining on single-cell suspensions prepared from day 11 EBs with or without tet treatment. Single cells prepared from both control (top panel) and Scl EBs (bottom panel) were stained with CD11b antibody, a marker for myeloid progenitors/macrophages. A high number of CD11b+ cells was seen from untreated EBs (wild-type and Scl) and day 4 tet-treated Scl EBs. Scale bar represents 100 μm. CAPP indicates parental vector stably transfected clones (wild-type); Scl, Scl stably transfected clones; Untx, untreated; TD0, tet-treated at day 0; TD2, tet-treated at day 2; TD4, tet-treated at day 4; CAPP-3, CAPP clone 3; Scl-10, Scl clone 10; Hb-α, hemoglobin-α. Both CAPP control and Scl clones are ZHBTc4-derived Oct-4 knockout ESCs with tet-regulated Oct-4 rescue.

Formation of hematopoietic progenitors from day 14 EBs derived from control and Scl ESCs. (A) Control and Scl ESCs cultured in methylcellulose medium supplemented with hematopoietic cytokines (stem cell factor, interleukin-1, interleukin-3, granulocyte-macrophage colony-stimulating factor, erythropoietin) in the absence of leukemia inhibiting factor. Scale bar represents 150 μm. (B) Immunoblot analysis of total protein extracts prepared from day 14 EBs derived from wild-type and Scl ESC clones. (Top panel) Immunoblotting with probe targeting hemoglobin-α antigen (1:1000). (Bottom panel) Immunoblotting with probe targeting β-actin antigen (1:10 000). Lanes 2 and 6 (TD0) were spliced from another blot because of the extremely low yield of EBs from TD0 ESCs. The results are consistent from separate experiments. (C) Representative histograms of flow cytometric analyses on 2 clones from wild-type ESCs and 2 clones from Scl ESCs. CD45 marker was analyzed by the FL4 channel (y-axis), and CD11b marker was analyzed by the FL2 channel (x-axis). (D) Calculation of the CD45CD11b double-positive cell populations from each clone. All cell preparations were done in duplicates, and each cell population was averaged from 2 separate experiments. Error bars represent 1 SD. (E) CD11b immunostaining on single-cell suspensions prepared from day 11 EBs with or without tet treatment. Single cells prepared from both control (top panel) and Scl EBs (bottom panel) were stained with CD11b antibody, a marker for myeloid progenitors/macrophages. A high number of CD11b+ cells was seen from untreated EBs (wild-type and Scl) and day 4 tet-treated Scl EBs. Scale bar represents 100 μm. CAPP indicates parental vector stably transfected clones (wild-type); Scl, Scl stably transfected clones; Untx, untreated; TD0, tet-treated at day 0; TD2, tet-treated at day 2; TD4, tet-treated at day 4; CAPP-3, CAPP clone 3; Scl-10, Scl clone 10; Hb-α, hemoglobin-α. Both CAPP control and Scl clones are ZHBTc4-derived Oct-4 knockout ESCs with tet-regulated Oct-4 rescue.

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