Figure 3
Figure 3. Increased number of cycling and undifferentiated erythroid cells in thalassemic versus healthy mice. Immunostaining for Ki-67 on spleen (A) and liver (B) specimens and for Mcm3 on liver (C) showed an increased number of cycling and undifferentiated cells in extramedullary sites of thalassemic mice (magnification, 400×). Mcm3 was also probed on spleen sections and the pattern was very similar to that observed for Ki-67 (data not shown). In particular, thalassemic liver sections showed an increased number of proliferating cells in areas associated with EMH. Cyclin-B1 staining (data not shown) confirmed that more proliferating cells are present in the spleens of thalassemic compared with wt mice. (D) Staining and analysis of cytospins of purified splenic erythroid cells after injection of BrdU in vivo showed that there is an increased percentage of cycling erythroid cells in β-thalassemic mice compared with healthy (20%, 30%, and 40% in wt, th3/+, and th3/th3, respectively; magnification, 400×). For panels A through D, images were captured on a Nikon Eclipse E800 microscope with a Retiga Exi camera (Qimaging) and a Plan Fluor 40×/0.75 numerical aperture objective, then acquired using the IPLab 3.65a software (Scanalytics). Brightness/contrast and color balance were adjusted using Abobe Photoshop 7.0.1 (Adobe Systems). (E) FACS analysis of CFSE-treated cells costained with antibodies to CD71 and Ter119. Erythroid cells from wt mice cultured in the presence of colcemid (purple line) or AG490 (blue line) showed little difference from untreated cells (pink line). Staining with 7-AAD, PI, and annexin-V excluded dead or apoptotic cells (n = 4 per genotype). After 48 hours, no further cell expansion was observed; instead there is a decline in cell number, indicating that these cells did not have an intrinsic self-sustaining ability to proliferate under these tissue culture conditions. (F) FACS analysis of freshly purified erythroid cells using an antibody that recognizes the phosphorylated form of Jak2 (green line). The blue line represents the cells stained with the isotype. As a control for the specificity of the antibody, the same cells were stained with the antibody after preincubation with the competitor peptide (red line, n = 3 per genotype).

Increased number of cycling and undifferentiated erythroid cells in thalassemic versus healthy mice. Immunostaining for Ki-67 on spleen (A) and liver (B) specimens and for Mcm3 on liver (C) showed an increased number of cycling and undifferentiated cells in extramedullary sites of thalassemic mice (magnification, 400×). Mcm3 was also probed on spleen sections and the pattern was very similar to that observed for Ki-67 (data not shown). In particular, thalassemic liver sections showed an increased number of proliferating cells in areas associated with EMH. Cyclin-B1 staining (data not shown) confirmed that more proliferating cells are present in the spleens of thalassemic compared with wt mice. (D) Staining and analysis of cytospins of purified splenic erythroid cells after injection of BrdU in vivo showed that there is an increased percentage of cycling erythroid cells in β-thalassemic mice compared with healthy (20%, 30%, and 40% in wt, th3/+, and th3/th3, respectively; magnification, 400×). For panels A through D, images were captured on a Nikon Eclipse E800 microscope with a Retiga Exi camera (Qimaging) and a Plan Fluor 40×/0.75 numerical aperture objective, then acquired using the IPLab 3.65a software (Scanalytics). Brightness/contrast and color balance were adjusted using Abobe Photoshop 7.0.1 (Adobe Systems). (E) FACS analysis of CFSE-treated cells costained with antibodies to CD71 and Ter119. Erythroid cells from wt mice cultured in the presence of colcemid (purple line) or AG490 (blue line) showed little difference from untreated cells (pink line). Staining with 7-AAD, PI, and annexin-V excluded dead or apoptotic cells (n = 4 per genotype). After 48 hours, no further cell expansion was observed; instead there is a decline in cell number, indicating that these cells did not have an intrinsic self-sustaining ability to proliferate under these tissue culture conditions. (F) FACS analysis of freshly purified erythroid cells using an antibody that recognizes the phosphorylated form of Jak2 (green line). The blue line represents the cells stained with the isotype. As a control for the specificity of the antibody, the same cells were stained with the antibody after preincubation with the competitor peptide (red line, n = 3 per genotype).

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