Identification of key regulatory pathways of myeloid differentiation using an mESC-based karyotypically normal cell model by Dong Li, Hong Yang, Hong Nan, Peng Liu, Sulei Pang, Qian Zhao, Rotem Karni, Mark P. Kamps, Yuanfu Xu, Jiaxi Zhou, Therese Wiedmer, Peter J. Sims, and Fei Wang Blood Volume 120(24):4712-4719 December 6, 2012 ©2012 by American Society of Hematology

Characterization of ESC-derived immortalized myeloid progenitors. Characterization of ESC-derived immortalized myeloid progenitors. (A) Wright-Giemsa staining of mEB8-ER (left) or mBB8-ER (right) cells. Bar represents 10 μm. (B) Surface expression of Sca-1, c-Kit, CD45, and CD41 in mEB8-ER and mBB8-ER cells assessed with flow cytometry. Nonspecific IgG was used as a negative control (denoted as “No staining”). (C) Analysis of EB8-ER cells using the CFU assay. Phase-contrast images of cells with (left) or without (right) β-estradiol treatment. (D) The relative percentage of GM, G, and M colonies formed from mEB8-ER cells in the absence of β-estradiol. Quantification of 4 separate experiments. Each represents the mean ± SEM (error bars). (C-D) Cells were cultivated in MethoCult GF M3434 medium for 10 days. Bar represents 50 μm. Dong Li et al. Blood 2012;120:4712-4719 ©2012 by American Society of Hematology

Differentiation of ESC-derived progenitors to neutrophils. Differentiation of ESC-derived progenitors to neutrophils. (A) Wright-Giemsa staining of mEB8-ER (top) or mBB8-ER cells (bottom) 6 days after the addition of 2 ng/mL G-CSF. (B) Surface expression of Gr-1, CD11b, CD16, and CD80 in mEB8-ER (top) and mBB8-ER cells (bottom) assessed with flow cytometry. Cells were induced to differentiate for 6 days. Cells before induction of differentiation (“0 day”) were used as a negative control (“Control”). (C) Analysis of intracellular [Ca2+] in mEB8-ER and mBB8-ER–derived neutrophils in response to fMLP stimulation. Fluorescence was recorded using SpectraMax M2 spectrometer (excitation, 340-380 nm; emission, 510 nm). Results are plotted as emission ratio versus time. Arrow indicates the time of addition of fMLP (100nM). A representative experiment from 5 separate experiments. (D) Migration in the transwell assay of mEB8-ER and mBB8-ER–derived neutrophils. Left: Representative images of cells that crossed the transwell membrane with or without the gradient of fMLP (100nM). Right: The count of cells that crossed the transwell membrane. Each bar represents the mean ± SEM (error bars). *P < .05. All values were normalized to the level (= 1) in cells without fMLP stimulation. Dong Li et al. Blood 2012;120:4712-4719 ©2012 by American Society of Hematology

Rapmycin disrupts G-CSF–induced myeloid differentiation of mEB8-ER/mBB8-ER cells. Rapmycin disrupts G-CSF–induced myeloid differentiation of mEB8-ER/mBB8-ER cells. (A-B) Top panel: Western blot of threonine-phosphorylated S6K (at residue 389: p-S6K) in mEB8-ER (A) or mBB8-ER (B) cells with or without rapamycin treatment. Cells were treated or not treated with G-CSF (2 ng, 30 minutes). Total S6K was a loading control. A typical experiment of 4 independent experiments. Bottom panel: Relative protein levels of p-S6K in mEB8-ER or mBB8-ER cells with various treatments. Quantification of blots from 4 separate experiments. Each bar represents the mean ± SEM. *P < .05. (C-D) Wright-Giemsa staining (top) and relative percentage of CD11b, Gr-1, CD16, and CD80-positive cells (bottom) in mEB8-ER (C) or mBB8-ER cells (D) measured with flow cytometry. Cells were induced to differentiate in the presence of G-CSF (2 ng/mL, 6 days) with (“G + Rap”) or without (“G-CSF”) rapamycin treatment (100nM). Four separate experiments were conducted, and quantification of 3 replicates of a typical experiment. Each bar represents the mean ± SEM (error bars). All values were normalized to the level (= 1) in cells without rapamycin treatment. *P < .05. Bar represents 10 μm. Dong Li et al. Blood 2012;120:4712-4719 ©2012 by American Society of Hematology

mTORC1, but not mTORC2, is necessary for myeloid differentiation of mEB8-ER cells. mTORC1, but not mTORC2, is necessary for myeloid differentiation of mEB8-ER cells. (A,C,E) Top panel: Western blot of mTOR (A), Raptor (C), or S6K1 (E) in mEB8-ER cells with or without shRNAs targeting mTOR, Raptor, or S6K1. α-tubulin was a loading control. A typical blot from 4 independent experiments. Bottom panel: Quantification of blots from 4 separate experiments. Each bar represents the mean ± SEM. *P < .05. LMP/pMKO: the empty retrovector. (B,D,F) Relative percentage of CD11b and Gr-1–positive cells with or without mTOR (B), Raptor (D), and S6K1 (F) depletion, measured with flow cytometry. Four separate experiments were conducted, and quantification of 3 replicates of a typical experiment. Each bar represents the mean ± SEM (error bars). All values were normalized to the level (= 1) in cells without depletion. *P < .05. Dong Li et al. Blood 2012;120:4712-4719 ©2012 by American Society of Hematology

Effects of mTOR inhibition in primary progenitors and in vivo. Effects of mTOR inhibition in primary progenitors and in vivo. (A) Surface expression of Sca-1 and c-Kit in mouse bone marrow progenitors (mBM) assessed by flow cytometry. c-Kit–positive and Sca-1–negative cells were sorted and used for myeloid differentiation. (B) Relative percentage of CD11b and Gr-1–positive cells in mouse bone marrow progenitors with or without rapamycin treatment (100nM), measured with flow cytometry. Cells were induced to differentiate in the presence of G-CSF (100 ng/mL, 10 days). Four separate experiments were conducted, and quantification of 3 replicates of a typical experiment are shown. Each bar represents the mean ± SEM (error bars). All values were normalized to the level (= 1) in cells without rapamycin treatment. *P < .05. (C) Relative percentage of CD11b and Gr-1–positive cells in mouse bone marrow progenitors with or without mTOR, Raptor, and S6K1 depletion, measured with flow cytometry. mTORshRNA1, Raptor shRNA1, and S6K1 shRNA were used to deplete mTOR, Raptor, and S6K1, respectively. NT (nontargeting) denotes cells infected with virus containing the empty retrovector (latent membrane protein or pMKO). Each bar represents the mean ± SEM (error bars) of 4 separate experiments. All values were normalized to the level (= 1) in cells without rapamycin treatment. *P < .05. (D) Relative percentage of lymphocytes in peripheral blood after 1-week rapamycin administration in vivo. “Control” and “Rap” indicate injection of vehicle and rapamycin, respectively. (E) Relative percentage of neutrophils and GMPs in the bone marrow after 1-week rapamycin administration in vivo. “Control” and “Rap” indicate injection of vehicle and rapamycin, respectively. (D-E) Three separate experiments were conducted, and in each experiment 10 mice were used. Each bar represents the mean ± SEM (error bars). All values were normalized to the level (= 100%) in cells without rapamycin treatment. *P < .05. Dong Li et al. Blood 2012;120:4712-4719 ©2012 by American Society of Hematology