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2.
Figure 6

Figure 6. Phenotypic, molecular, and functional analysis of H1 hESC–derived osteoclasts.. From: Generation of mature human myelomonocytic cells through expansion and differentiation of pluripotent stem cell-derived lin–CD34+CD43+CD45+ progenitors .

(A) FACS analysis of hESC-derived osteoclasts. A representative of 3 independent experiments is shown. (B) RT-PCR analysis of gene expression in hESC-derived osteoclasts. Analysis of mineralized matrix–resorptive capacity of hESC-derived macrophages (C and D) and osteoclasts (E and F) using BioCoat osteologic discs. Resorption lacunae around multinucleated cells are clearly visible in osteoclasts cultures (E, inset) but not in cells cultured with M-CSF and IL-1β (C). Visualization of resorption lacunae in macrophage (D) and osteoclast (F) cultures with von Kossa staining. Scale bar: 200 μm (CF).

Kyung-Dal Choi, et al. J Clin Invest. 2009 September 1;119(9):2818-2829.
3.
Figure 3

Figure 3. Morphology and cytochemical features of H1 hESC–derived myeloid progenitors and differentiated myelomonocytic cells.. From: Generation of mature human myelomonocytic cells through expansion and differentiation of pluripotent stem cell-derived lin–CD34+CD43+CD45+ progenitors .

(A) Wright-stained cytospins of H1-derived CD235a/CD41aCD45+ cells after 2 days expansion with GM-CSF (scale bar: 10 μm). (B) Typical GEMM-CFC generated from day 2 GM-CSF–expanded cells (scale bar: 200 μm). Wright-stained cytospins of GEMM-CFC (scale bar: 40 μm) (C), hESC-derived mature neutrophils (scale bar: 25 μm; inset, 5 μm) (D), eosinophils (scale bar: 40 μm; inset, 10 μm) (E), CD1a+ cells isolated from DC (scale bar: 10 μm) (G) and LC (scale bar: 10 μm) (H) cultures, and macrophages (scale bar: 100 μm; inset, 20 μm) (I). (F) Cytochemical staining for eosinophil-specific (cyanide-resistant) peroxidase of hESC-derived eosinophils (scale bar: 50 μm; inset, 10 μm). (J) Cytochemical staining for TRAP of hESC-derived osteoclasts (scale bar: 300 μm; inset, 100 μm). Phase-contrast (K) and corresponding DAPI staining (L) of osteoclasts to demonstrate multinucleation (scale bar: 200 μm).

Kyung-Dal Choi, et al. J Clin Invest. 2009 September 1;119(9):2818-2829.
4.
Figure 5

Figure 5. Phenotypic, molecular, and functional analysis of H1 hESC–derived DCs, LCs, and macrophages obtained from isolated CD235a/CD41a–CD45+ cells after 2 days expansion with GM-CSF. . From: Generation of mature human myelomonocytic cells through expansion and differentiation of pluripotent stem cell-derived lin–CD34+CD43+CD45+ progenitors .

FACS analysis of hESC-derived DCs (A), LCs (B), and macrophages (C). Plots show isotype control (gray) and specific antibody (black) histograms; asterisks indicate intracellular staining. Values within dot plots indicate the percentage of cells within the corresponding gate. A representative of 5–10 independent experiments is shown. (D) FACS analysis of OVA uptake and processing by hESC-derived antigen-presenting cells. Plots show isotype control (open gray), cells incubated at 4°C (filled gray; nonspecific binding control), and 37°C (open black) histograms. (E) Allogeneic stimulatory capacity of hESC-derived antigen-presenting cells. PB, peripheral blood lymphocytes; CB, cord blood lymphocytes. Results are mean ± SEM of 3 independent experiments performed in triplicate. (F) RT-PCR analysis of gene expression in hESC-derived antigen-presenting cells. Mϕ, macrophages; OC, osteoclasts.

Kyung-Dal Choi, et al. J Clin Invest. 2009 September 1;119(9):2818-2829.
5.
Figure 2

Figure 2. Characterization of H1 hESC–derived myeloid progenitors generated in OP9 coculture and expanded with GM-CSF.. From: Generation of mature human myelomonocytic cells through expansion and differentiation of pluripotent stem cell-derived lin–CD34+CD43+CD45+ progenitors .

Phenotype of CD235a/CD41aCD45+ cells generated on day 9 (d9) of hESC/OP9 coculture (A) and after 2 (B) and 8 days (C) expansion with GM-CSF; the asterisk indicates intracellular staining. A representative of 3–5 independent experiments is shown. SSC, side scatter. Values within dot plots indicate the percentage of cells in the respective quadrants. (D) Absolute number of myeloid CFCs generated from one 10-cm dish of hESC/OP9 coculture and from CD235a/CD41aCD45+ cells obtained from the same dish and expanded in the presence of GM-CSF for 2–8 days. Results are mean ± SEM of 3–5 independent experiments. (E) Kinetics of CD235a/CD41aCD45+ cell expansion with GM-CSF. Data are expressed as the number of CD235a/CD41aCD45+ cells generated from one 10-cm dish of hESC/OP9 coculture and following expansion with GM-CSF. Results are mean ± SEM of 3–5 experiments. (F) Typical morphology of granulocytic colony obtained from CD235a/CD41aCD45+ cells expanded with GM-CSF for 2 and 6 days (scale bars: 100 μm). The graph shows cell numbers per single granulocytic colony obtained from cells expanded with GM-CSF for 2–8 days.

Kyung-Dal Choi, et al. J Clin Invest. 2009 September 1;119(9):2818-2829.
6.
Figure 4

Figure 4. Phenotypic, molecular, and functional analysis of H1 hESC-derived eosinophils and neutrophils obtained from isolated CD235a/CD41a–CD45+ cells after 2 days expansion with GM-CSF. . From: Generation of mature human myelomonocytic cells through expansion and differentiation of pluripotent stem cell-derived lin–CD34+CD43+CD45+ progenitors .

FACS analysis of phenotype of hESC-derived neutrophils (A) and eosinophils (B). Plots show isotype control (gray) and specific antibody (black) histograms; asterisks indicate intracellular staining. Values within dot plots indicate the percentage of cells within the corresponding gate. A representative of 10 independent experiments is shown. Analysis of neutrophil and eosinophil-specific gene expression in hESC-derived neutrophils (C) and eosinophils (D) by RT-PCR. M, molecular weight markers; hES, undifferentiated hESCs; 45+, hESC-derived CD235a/CD41aCD45+ cells isolated after 2 days expansion with GM-CSF; Neu, neutrophils; Eo is eosinophils. (E) Phagocytosis of zymozan and E. coli particles by hESC-derived neutrophils. Plots show histograms for isotype control (open gray) and cells incubated at 4°C (filled gray; nonspecific binding control) and 37°C (open black). (F) Superoxide production by hESC-derived (H1-Neu), somatic CD34+ cell–derived (34-Neu), and peripheral blood (PB-Neu) neutrophils in response to PMA. Results are mean ± SEM of 3 independent experiments performed in triplicate; **P < 0.01. (G) Superoxide production by hESC-derived (H1-EO) and somatic CD34+ cell–derived (34-EO) eosinophils in response to PMA. Results are mean ± SEM of 3 independent experiments performed in triplicate. (H) Chemotactic activity of hESC-derived, somatic CD34+ cell–derived, and peripheral blood neutrophils. Results are mean ± SEM of 3 independent experiments performed in triplicate; *P < 0.05. (I) Chemotactic activity of hESC-derived and somatic CD34+ cell–derived eosinophils. Results are mean ± SEM of 3 independent experiments performed in triplicate; **P < 0.01.

Kyung-Dal Choi, et al. J Clin Invest. 2009 September 1;119(9):2818-2829.

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