Figure 1
Hes1-transduced KSLs, CMPs, or GMPs were immortalized in the presence of IL-3. (A) Typical colonies derived from Hes1- and empty vector-transduced KSLs, CMPs, and GMPs in the presence of SCF, TPO, IL-3, and IL-6. Images were obtained with an IX70 microscope and a DP70 camera (Olympus); an objective lens, UPlanFl (Olympus); original magnification ×40 (bottom 2 in the right panels) and original magnification ×100 (remaining 4 panels). (B) Giemsa staining of Hes1- and control vector-transduced KSLs, CMPs, and GMPs. Images were obtained with a BX51 microscope and a DP12 camera (Olympus); an objective lens, UPlanFl (Olympus); original magnification ×1000. (C) Colony-forming assay from KSLs, CMPs, and GMPs transduced with Hes1 or empty vector. Hes1-transduced cells were replatable more than 4 times in vitro. Bars represent the number of colonies obtained per 103 cells after each round of plating in methylcellulose supplemented with SCF, TPO, IL-3, and IL-6. A representative result from 3 independent and reproducible experiments is shown. Error bars represent the SD from duplicate cultures. (D) Sustained growth of Hes1-transduced cells in liquid culture supplemented with 1 ng/mL IL-3. The number of cells was determined every 7 days by trypan blue staining, and 105 cells per well were seeded into a 6-well plate. Liquid culture was reproducibly continued for more than 6 months. (E) Cytokine requirement of Hes1-transduced CMPs. The cells were cultured in Iscove modified Dulbecco medium supplemented with indicated cytokines in duplicate. The numbers of cells were counted after 4 days of culture. A representative result from 2 independent and reproducible experiments is shown. Error bars represent the SD from duplicate cultures. Hes1-transduced KSLs and GMPs showed similar results (supplemental Figure 1A-B). (F) Flow-cytometric analysis of Hes1-transduced KSLs cultured in methylcellulose supplemented with SCF, TPO, IL-3, and IL-6. The dot plots represent Gr-1, CD19, c-Kit, CD4, CD8a, B220, Sca-1, CD34, Ter119, and CD14 labeled with a corresponding PE-conjugated monoclonal antibody versus CD11b, CD3, and FcϵR1α labeled with a corresponding fluorescein isothiocyanate-conjugated monoclonal antibody or FL1 with no monoclonal antibody. Hes1-transduced CMPs and GMPs showed similar expression patterns (supplemental Figure 2A-B). (G) Flow-cytometric analysis of Lin−-gated Hes1-transduced cells. Five-color analyses are used to identify KSL-like (top panels) and CMP-like and GMP-like cells (bottom panels) in the Hes1-transduced KSLs, CMPs, and GMPs. The number shows the percentage of cells in all nucleated cells. The analyzed cells were NGFR sorted at 48 to 60 hours from the initiation of Hes1- or control vector-transduction and cultured for the following lengths of time before the analysis: (A) 1 week, (B) 1 week, (C) 0 days, (D) 4 weeks, (E) 2 weeks, (F) 1 week, and (E) 2 weeks.

Hes1-transduced KSLs, CMPs, or GMPs were immortalized in the presence of IL-3. (A) Typical colonies derived from Hes1- and empty vector-transduced KSLs, CMPs, and GMPs in the presence of SCF, TPO, IL-3, and IL-6. Images were obtained with an IX70 microscope and a DP70 camera (Olympus); an objective lens, UPlanFl (Olympus); original magnification ×40 (bottom 2 in the right panels) and original magnification ×100 (remaining 4 panels). (B) Giemsa staining of Hes1- and control vector-transduced KSLs, CMPs, and GMPs. Images were obtained with a BX51 microscope and a DP12 camera (Olympus); an objective lens, UPlanFl (Olympus); original magnification ×1000. (C) Colony-forming assay from KSLs, CMPs, and GMPs transduced with Hes1 or empty vector. Hes1-transduced cells were replatable more than 4 times in vitro. Bars represent the number of colonies obtained per 103 cells after each round of plating in methylcellulose supplemented with SCF, TPO, IL-3, and IL-6. A representative result from 3 independent and reproducible experiments is shown. Error bars represent the SD from duplicate cultures. (D) Sustained growth of Hes1-transduced cells in liquid culture supplemented with 1 ng/mL IL-3. The number of cells was determined every 7 days by trypan blue staining, and 105 cells per well were seeded into a 6-well plate. Liquid culture was reproducibly continued for more than 6 months. (E) Cytokine requirement of Hes1-transduced CMPs. The cells were cultured in Iscove modified Dulbecco medium supplemented with indicated cytokines in duplicate. The numbers of cells were counted after 4 days of culture. A representative result from 2 independent and reproducible experiments is shown. Error bars represent the SD from duplicate cultures. Hes1-transduced KSLs and GMPs showed similar results (supplemental Figure 1A-B). (F) Flow-cytometric analysis of Hes1-transduced KSLs cultured in methylcellulose supplemented with SCF, TPO, IL-3, and IL-6. The dot plots represent Gr-1, CD19, c-Kit, CD4, CD8a, B220, Sca-1, CD34, Ter119, and CD14 labeled with a corresponding PE-conjugated monoclonal antibody versus CD11b, CD3, and FcϵR1α labeled with a corresponding fluorescein isothiocyanate-conjugated monoclonal antibody or FL1 with no monoclonal antibody. Hes1-transduced CMPs and GMPs showed similar expression patterns (supplemental Figure 2A-B). (G) Flow-cytometric analysis of Lin−-gated Hes1-transduced cells. Five-color analyses are used to identify KSL-like (top panels) and CMP-like and GMP-like cells (bottom panels) in the Hes1-transduced KSLs, CMPs, and GMPs. The number shows the percentage of cells in all nucleated cells. The analyzed cells were NGFR sorted at 48 to 60 hours from the initiation of Hes1- or control vector-transduction and cultured for the following lengths of time before the analysis: (A) 1 week, (B) 1 week, (C) 0 days, (D) 4 weeks, (E) 2 weeks, (F) 1 week, and (E) 2 weeks.

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