Figure 4
Figure 4. TPO changes the activation status of β3-integrin through inside-out signaling, and post-ligation outside-in signaling via β3PY747 is indispensable for maintenance of HSC function during ex vivo expansion. (A) CD34−KSL cells derived from WT and β3−/− mice were cultured with AlexaFlour 647–labeled fibrinogen in S-Clone SF-03 medium, with or without SCF or TPO. The fluorescence intensity of the bound fibrinogen was analyzed by flow cytometry: white represents no cytokine; and gray, stimulation of cytokine. The graphs represent the relative mean fluorescence intensity (MFI); binding in the absence of cytokine served as the control. Data are mean ± SD; n > 3. *P < .01. (B-C) Forty CD34−KSL cells obtained from BM of L746A (Ly5.2; B) or Y747A mice (Ly5.2; C) were cultured with TPO for 5 days in the presence of 2C9.G2 or IgG and examined using transplantation assays, as described in Figure 2. To exogenously induce integrin activation (change the structure to the activated state), Mn2+ was added to TPO-containing medium. (D) After culture of 1000 sorted L746A-mutant CD34−KSL cells (Ly5.1) for 5 days with 2C9.G2 or hamster IgG (isotype control) in the presence of TPO and Mn2+, the percentages of KSL and CD48−KSL cells were determined by flow cytometry. The values in the dot plots are mean ± SD. (E) After the culture, the total cell number was counted. The graph represents the fold increase in total cell number after 5 days of culture. Data are mean ± SD. (F) Forty WT CD34−KSL cells (Ly5.1) were also cultured for 5 days in medium containing SCF and Mn2+ along with 2C9.G2 or IgG. (G) Using plates coated with VN, OPN, or BSA, 40 WT CD34−KSL cells (Ly5.1) were cultured with TPO for 5 days in the absence or presence of Mn2+. After the culture, cells were transplanted along with 2 × 105 BM competitor cells into irradiated recipient mice. The plots represent the percentage of donor (Ly5.2 or Ly5.1)-derived cells in the peripheral blood of individual mice 12 weeks after transplantation. *P < .01. **P < .05. Recipient mice with donor cell chimerism of < 1.0% for any lineage were considered not to be reconstituted (negative mice).

TPO changes the activation status of β3-integrin through inside-out signaling, and post-ligation outside-in signaling via β3PY747 is indispensable for maintenance of HSC function during ex vivo expansion. (A) CD34−KSL cells derived from WT and β3−/− mice were cultured with AlexaFlour 647–labeled fibrinogen in S-Clone SF-03 medium, with or without SCF or TPO. The fluorescence intensity of the bound fibrinogen was analyzed by flow cytometry: white represents no cytokine; and gray, stimulation of cytokine. The graphs represent the relative mean fluorescence intensity (MFI); binding in the absence of cytokine served as the control. Data are mean ± SD; n > 3. *P < .01. (B-C) Forty CD34−KSL cells obtained from BM of L746A (Ly5.2; B) or Y747A mice (Ly5.2; C) were cultured with TPO for 5 days in the presence of 2C9.G2 or IgG and examined using transplantation assays, as described in Figure 2. To exogenously induce integrin activation (change the structure to the activated state), Mn2+ was added to TPO-containing medium. (D) After culture of 1000 sorted L746A-mutant CD34−KSL cells (Ly5.1) for 5 days with 2C9.G2 or hamster IgG (isotype control) in the presence of TPO and Mn2+, the percentages of KSL and CD48−KSL cells were determined by flow cytometry. The values in the dot plots are mean ± SD. (E) After the culture, the total cell number was counted. The graph represents the fold increase in total cell number after 5 days of culture. Data are mean ± SD. (F) Forty WT CD34−KSL cells (Ly5.1) were also cultured for 5 days in medium containing SCF and Mn2+ along with 2C9.G2 or IgG. (G) Using plates coated with VN, OPN, or BSA, 40 WT CD34−KSL cells (Ly5.1) were cultured with TPO for 5 days in the absence or presence of Mn2+. After the culture, cells were transplanted along with 2 × 105 BM competitor cells into irradiated recipient mice. The plots represent the percentage of donor (Ly5.2 or Ly5.1)-derived cells in the peripheral blood of individual mice 12 weeks after transplantation. *P < .01. **P < .05. Recipient mice with donor cell chimerism of < 1.0% for any lineage were considered not to be reconstituted (negative mice).

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