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Bmi-1 Regulates Extensive Erythroid Self-Renewal.

Kim AR, Olsen JL, England SJ, Huang YS, Fegan KH, Delgadillo LF, McGrath KE, Kingsley PD, Waugh RE, Palis J - Stem Cell Reports (2015)

Bottom Line: To better understand the mechanisms regulating extensive erythroid self-renewal, global gene expression data sets from self-renewing and differentiating erythroblasts were analyzed and revealed the differential expression of Bmi-1.Bmi-1 overexpression conferred extensive self-renewal capacity upon adult bone-marrow-derived self-renewing erythroblasts, which normally have limited proliferative potential.Importantly, Bmi-1 transduction did not interfere with the ability of extensively self-renewing erythroblasts (ESREs) to terminally mature either in vitro or in vivo.

View Article: PubMed Central - PubMed

Affiliation: Center for Pediatric Biomedical Research, Department of Pediatrics, University of Rochester Medical Center, Rochester, NY 14642, USA; Department of Biomedical Genetics, University of Rochester Medical Center, Rochester, NY 14642, USA.

No MeSH data available.


Related in: MedlinePlus

Intravenous Transfusion of iESREs Results in a Transient Wave of Reticulocytes and the Circulation of iESRE-Derived RBCs for Several Weeks(A) Kinetics of GFP+/CD71+ reticulocytes (left panel) and GFP+/CD71− mature RBCs derived from 10 × 107 GFP+ iESREs transfused on day 0 into recipient mice. The percentages of iESRE-derived reticulocytes and RBCs were calculated as the number of GFP+ cells divided by the total cell number in the parent gate (mean ± SEM; N = 3 mice).(B) The morphology of circulating mature RBCs (Ter119+/CD71−/DRAQ5−), was visualized by imaging flow cytometry on day 6 post-GFP+-iESRE transfusion. Reticulocytes and RBCs derived from iESREs (GFP+, upper panel) showed similar biconcave shape as endogenous (GFP−, lower panel) RBCs. Representative data from one of three mice are shown (size bar represents 10 μm).(C) The size of iESRE-derived (GFP+) and endogenous (GFP−) circulating reticulocytes and RBCs on day 6 post-transfusion were compared by imaging flow cytometry. Reticulocytes were similar in size (mean ± SEM; N = 3 independent experiments; p = 0.22; left panel), while iESRE-derived RBCs were significantly larger than endogenous RBCs (mean ± SEM; N = 3 independent experiments; right panel). p value was calculated using two-tailed Student’s t test. ∗p < 0.05.(D) Fluorescence-imaged microdeformation analysis of iESRE-derived (GFP+, upper panel) and endogenous (GFP−, lower panel) RBCs.(E) Sphericity of iESRE-derived (GFP+) and endogenous (GFP−) RBCs on day 26 post-transfusion, based on micropipette measurements of surface area and volume measurements (individual value ± SD; p value = 0.177). p value was calculated using two-tailed Student’s t test. Representative data from one of three mice are shown.See also Figure S3.
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fig4: Intravenous Transfusion of iESREs Results in a Transient Wave of Reticulocytes and the Circulation of iESRE-Derived RBCs for Several Weeks(A) Kinetics of GFP+/CD71+ reticulocytes (left panel) and GFP+/CD71− mature RBCs derived from 10 × 107 GFP+ iESREs transfused on day 0 into recipient mice. The percentages of iESRE-derived reticulocytes and RBCs were calculated as the number of GFP+ cells divided by the total cell number in the parent gate (mean ± SEM; N = 3 mice).(B) The morphology of circulating mature RBCs (Ter119+/CD71−/DRAQ5−), was visualized by imaging flow cytometry on day 6 post-GFP+-iESRE transfusion. Reticulocytes and RBCs derived from iESREs (GFP+, upper panel) showed similar biconcave shape as endogenous (GFP−, lower panel) RBCs. Representative data from one of three mice are shown (size bar represents 10 μm).(C) The size of iESRE-derived (GFP+) and endogenous (GFP−) circulating reticulocytes and RBCs on day 6 post-transfusion were compared by imaging flow cytometry. Reticulocytes were similar in size (mean ± SEM; N = 3 independent experiments; p = 0.22; left panel), while iESRE-derived RBCs were significantly larger than endogenous RBCs (mean ± SEM; N = 3 independent experiments; right panel). p value was calculated using two-tailed Student’s t test. ∗p < 0.05.(D) Fluorescence-imaged microdeformation analysis of iESRE-derived (GFP+, upper panel) and endogenous (GFP−, lower panel) RBCs.(E) Sphericity of iESRE-derived (GFP+) and endogenous (GFP−) RBCs on day 26 post-transfusion, based on micropipette measurements of surface area and volume measurements (individual value ± SD; p value = 0.177). p value was calculated using two-tailed Student’s t test. Representative data from one of three mice are shown.See also Figure S3.

Mentions: We next asked whether iESREs also have the capacity to mature in vivo. To track erythrocytes after transfusion, iESREs were generated from the marrow of adult ubiquitin C (UBC)-GFP mice that express GFP driven by the ubiquitin C promoter. 5–10 × 107 iESREs were injected intravenously into NOD scid gamma (NSG) or C57BL/6J mice treated with 1.5 Gy total body irradiation (TBI) to transiently suppress endogenous erythropoiesis. While most ESREs were localized to the spleen and bone marrow 1 day after transfusion (Figure S3B), a small percentage of GFP+ reticulocytes were detected in the circulation, likely derived from the small number of spontaneously maturing (benzidine+) cells present in the self-renewing cultures (Figure 3A). A large, transient wave of reticulocytes entered the bloodstream between 4 and 8 days after iESRE transfusion into irradiated mice (Figures 4A and S3C). At day 6, 40%–90% of the reticulocytes in the bloodstream were GFP+ (Figures 4A and S3C).


Bmi-1 Regulates Extensive Erythroid Self-Renewal.

Kim AR, Olsen JL, England SJ, Huang YS, Fegan KH, Delgadillo LF, McGrath KE, Kingsley PD, Waugh RE, Palis J - Stem Cell Reports (2015)

Intravenous Transfusion of iESREs Results in a Transient Wave of Reticulocytes and the Circulation of iESRE-Derived RBCs for Several Weeks(A) Kinetics of GFP+/CD71+ reticulocytes (left panel) and GFP+/CD71− mature RBCs derived from 10 × 107 GFP+ iESREs transfused on day 0 into recipient mice. The percentages of iESRE-derived reticulocytes and RBCs were calculated as the number of GFP+ cells divided by the total cell number in the parent gate (mean ± SEM; N = 3 mice).(B) The morphology of circulating mature RBCs (Ter119+/CD71−/DRAQ5−), was visualized by imaging flow cytometry on day 6 post-GFP+-iESRE transfusion. Reticulocytes and RBCs derived from iESREs (GFP+, upper panel) showed similar biconcave shape as endogenous (GFP−, lower panel) RBCs. Representative data from one of three mice are shown (size bar represents 10 μm).(C) The size of iESRE-derived (GFP+) and endogenous (GFP−) circulating reticulocytes and RBCs on day 6 post-transfusion were compared by imaging flow cytometry. Reticulocytes were similar in size (mean ± SEM; N = 3 independent experiments; p = 0.22; left panel), while iESRE-derived RBCs were significantly larger than endogenous RBCs (mean ± SEM; N = 3 independent experiments; right panel). p value was calculated using two-tailed Student’s t test. ∗p < 0.05.(D) Fluorescence-imaged microdeformation analysis of iESRE-derived (GFP+, upper panel) and endogenous (GFP−, lower panel) RBCs.(E) Sphericity of iESRE-derived (GFP+) and endogenous (GFP−) RBCs on day 26 post-transfusion, based on micropipette measurements of surface area and volume measurements (individual value ± SD; p value = 0.177). p value was calculated using two-tailed Student’s t test. Representative data from one of three mice are shown.See also Figure S3.
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fig4: Intravenous Transfusion of iESREs Results in a Transient Wave of Reticulocytes and the Circulation of iESRE-Derived RBCs for Several Weeks(A) Kinetics of GFP+/CD71+ reticulocytes (left panel) and GFP+/CD71− mature RBCs derived from 10 × 107 GFP+ iESREs transfused on day 0 into recipient mice. The percentages of iESRE-derived reticulocytes and RBCs were calculated as the number of GFP+ cells divided by the total cell number in the parent gate (mean ± SEM; N = 3 mice).(B) The morphology of circulating mature RBCs (Ter119+/CD71−/DRAQ5−), was visualized by imaging flow cytometry on day 6 post-GFP+-iESRE transfusion. Reticulocytes and RBCs derived from iESREs (GFP+, upper panel) showed similar biconcave shape as endogenous (GFP−, lower panel) RBCs. Representative data from one of three mice are shown (size bar represents 10 μm).(C) The size of iESRE-derived (GFP+) and endogenous (GFP−) circulating reticulocytes and RBCs on day 6 post-transfusion were compared by imaging flow cytometry. Reticulocytes were similar in size (mean ± SEM; N = 3 independent experiments; p = 0.22; left panel), while iESRE-derived RBCs were significantly larger than endogenous RBCs (mean ± SEM; N = 3 independent experiments; right panel). p value was calculated using two-tailed Student’s t test. ∗p < 0.05.(D) Fluorescence-imaged microdeformation analysis of iESRE-derived (GFP+, upper panel) and endogenous (GFP−, lower panel) RBCs.(E) Sphericity of iESRE-derived (GFP+) and endogenous (GFP−) RBCs on day 26 post-transfusion, based on micropipette measurements of surface area and volume measurements (individual value ± SD; p value = 0.177). p value was calculated using two-tailed Student’s t test. Representative data from one of three mice are shown.See also Figure S3.
Mentions: We next asked whether iESREs also have the capacity to mature in vivo. To track erythrocytes after transfusion, iESREs were generated from the marrow of adult ubiquitin C (UBC)-GFP mice that express GFP driven by the ubiquitin C promoter. 5–10 × 107 iESREs were injected intravenously into NOD scid gamma (NSG) or C57BL/6J mice treated with 1.5 Gy total body irradiation (TBI) to transiently suppress endogenous erythropoiesis. While most ESREs were localized to the spleen and bone marrow 1 day after transfusion (Figure S3B), a small percentage of GFP+ reticulocytes were detected in the circulation, likely derived from the small number of spontaneously maturing (benzidine+) cells present in the self-renewing cultures (Figure 3A). A large, transient wave of reticulocytes entered the bloodstream between 4 and 8 days after iESRE transfusion into irradiated mice (Figures 4A and S3C). At day 6, 40%–90% of the reticulocytes in the bloodstream were GFP+ (Figures 4A and S3C).

Bottom Line: To better understand the mechanisms regulating extensive erythroid self-renewal, global gene expression data sets from self-renewing and differentiating erythroblasts were analyzed and revealed the differential expression of Bmi-1.Bmi-1 overexpression conferred extensive self-renewal capacity upon adult bone-marrow-derived self-renewing erythroblasts, which normally have limited proliferative potential.Importantly, Bmi-1 transduction did not interfere with the ability of extensively self-renewing erythroblasts (ESREs) to terminally mature either in vitro or in vivo.

View Article: PubMed Central - PubMed

Affiliation: Center for Pediatric Biomedical Research, Department of Pediatrics, University of Rochester Medical Center, Rochester, NY 14642, USA; Department of Biomedical Genetics, University of Rochester Medical Center, Rochester, NY 14642, USA.

No MeSH data available.


Related in: MedlinePlus