American Society of Hematology

Figure 6

$Figure 6. The role of MAPK pathway in lineage conversion and myeloid differentiation. (A) The IL-2RβdH mutant, which has the complete A region with H region removed, was introduced to CLPs. Double-sorted CLPs were first cultured with retrovirus containing supernatant in the presence of SCF, Flt3L, IL-7, and 5 μM PD98059 or 5 μM U0126 for 24 hours. IL-2 was then added into culture to induce lineage conversion, and cells were further cultured for an additional 24 hours. GFP+ cells were sorted onto methylcellulose medium and cultured in the presence of IL-3 and GM-CSF. Plating efficiency was determined by enumerating GM colonies after 5 days of culture. (B) To determine the B-cell developmental potential, CLPs were cultured with retroviral supernatants in the presence of SCF, Flt3L, IL-7, and MEK/ERK inhibitors for 48 hours. GFP+ cells were sorted and cocultured on an OP9 stromal layer with SCF, Flt3L and IL-7. B220+CD19+ B-cell readout was examined by FACS analysis at day 7. (C) CMPs were cultured in the presence of GM-CSF, IL-3, and SCF with MEK/ERK inhibitors as described for panel B. Plating efficiency was determined by counting GM colonies after 5 days of culture. (D) CMPs were sorted and cultured on OP9 stromal layers in the presence of MEK/ERK inhibitors. Fresh medium with inhibitors was added every 3 days during the culture period. The presence of Mac-1+Gr-1+ myeloid cells was examined by FACS analysis. (E) MEK/ERK activation is crucial in myeloid commitment from HSCs. Flt3−c-KithiLin−/loSca-1+ HSCs were sorted and cultured in the presence of MEK/ERK inhibitors as described. GM colony forming ability of HSCs was examined by enumerating colonies in methylcellulose medium supplemented with IL-3 and GM-CSF at day 6 of the culture. (F) Inhibition of MAPK activity represses myeloid cell readout from MPPs. The presence of both Mac-1+ and B220+ cells from single VCAM-1−Flt3+ MPPs was defined as bipotent (GM + B; red), whereas readout of either Mac-1+ or B220+ was defined as single lineage, GM only (blue), or B only (open). Bipotent and single lineage readout was examined in the absence (control) or presence of MEK/ERK inhibitors (PD98059 and U0126). (G) Loss-of-function and gain-of-function effect of MEK1 on lineage decision. HSCs that had been transduced with siRNA targeting MEK1 or with a constitutively active form of MEK1 were injected into RAG2−/− hosts. Peripheral blood was collected at week 4 and percentage of GM (Mac-1+) and B cells (B220+) in the GFP+ donor fraction was analyzed. T cells were under the detectable level in the blood at the time points we examined (2-6 weeks after injection). However, we could identify similar numbers of TCRβ+ T cells in the spleen at week 6 (data not shown). (A,C,E) Error bars represent standard deviation from triplicate samples. (B,D) Percentages indicate total cells in the gates as described in Figure 1C. (G) Horizontal bars represent the mean value from 3 mice.$

The role of MAPK pathway in lineage conversion and myeloid differentiation. (A) The IL-2RβdH mutant, which has the complete A region with H region removed, was introduced to CLPs. Double-sorted CLPs were first cultured with retrovirus containing supernatant in the presence of SCF, Flt3L, IL-7, and 5 μM PD98059 or 5 μM U0126 for 24 hours. IL-2 was then added into culture to induce lineage conversion, and cells were further cultured for an additional 24 hours. GFP⁺ cells were sorted onto methylcellulose medium and cultured in the presence of IL-3 and GM-CSF. Plating efficiency was determined by enumerating GM colonies after 5 days of culture. (B) To determine the B-cell developmental potential, CLPs were cultured with retroviral supernatants in the presence of SCF, Flt3L, IL-7, and MEK/ERK inhibitors for 48 hours. GFP⁺ cells were sorted and cocultured on an OP9 stromal layer with SCF, Flt3L and IL-7. B220⁺CD19⁺ B-cell readout was examined by FACS analysis at day 7. (C) CMPs were cultured in the presence of GM-CSF, IL-3, and SCF with MEK/ERK inhibitors as described for panel B. Plating efficiency was determined by counting GM colonies after 5 days of culture. (D) CMPs were sorted and cultured on OP9 stromal layers in the presence of MEK/ERK inhibitors. Fresh medium with inhibitors was added every 3 days during the culture period. The presence of Mac-1⁺Gr-1⁺ myeloid cells was examined by FACS analysis. (E) MEK/ERK activation is crucial in myeloid commitment from HSCs. Flt3⁻c-Kit^hiLin^−/loSca-1⁺ HSCs were sorted and cultured in the presence of MEK/ERK inhibitors as described. GM colony forming ability of HSCs was examined by enumerating colonies in methylcellulose medium supplemented with IL-3 and GM-CSF at day 6 of the culture. (F) Inhibition of MAPK activity represses myeloid cell readout from MPPs. The presence of both Mac-1⁺ and B220⁺ cells from single VCAM-1⁻Flt3⁺ MPPs was defined as bipotent (GM + B; red), whereas readout of either Mac-1⁺ or B220⁺ was defined as single lineage, GM only (blue), or B only (open). Bipotent and single lineage readout was examined in the absence (control) or presence of MEK/ERK inhibitors (PD98059 and U0126). (G) Loss-of-function and gain-of-function effect of MEK1 on lineage decision. HSCs that had been transduced with siRNA targeting MEK1 or with a constitutively active form of MEK1 were injected into RAG2^−/− hosts. Peripheral blood was collected at week 4 and percentage of GM (Mac-1⁺) and B cells (B220⁺) in the GFP⁺ donor fraction was analyzed. T cells were under the detectable level in the blood at the time points we examined (2-6 weeks after injection). However, we could identify similar numbers of TCRβ⁺ T cells in the spleen at week 6 (data not shown). (A,C,E) Error bars represent standard deviation from triplicate samples. (B,D) Percentages indicate total cells in the gates as described in Figure 1C. (G) Horizontal bars represent the mean value from 3 mice.

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