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Abstract
Background:
The Polycomb-group gene Bmi-1 is known to be a molecular regulator of self-renewal of normal and leukemic stem cells and be involved in various aspects of cellular proliferation, differentiation, and survival.
Methods:
This study evaluated the effects of overexpression of Bmi-1 on human cord blood CD34+ cells. Bmi-1 was introduced into CD34+ cells through lentivirus transduction. Bmi-1 expressing CD34+ cells were applied to colony forming assay, stromal co-culture, and cytokine-stimulatied culture.
Results:
Ectopic expression of Bmi-1 resulted in the increased number of erythroid colonies in primary and secondary colony forming assay in an erythropoietin dependent manner. In stromal co-culture, Bmi-1-expressing postnatal hematopoietic stem cells seemed to lose the ability of self-renewal, as determined by week 5 cobblestone area-forming cell assay and by week 5 secondary colony assay. In cytokine-stimulated suspension culture of Bmi-1-transduced CD34+ cells, we observed increased erythropoiesis marked by Glycophorin A expression.
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Fig. 1
Construction of FUEG and FUEG-Bmi-1 lentivirus and detection of elevated Bmi-1 expression in transduced 293T and human cord blood (CB) CD34+ cells. (A) A schematic image of FUEG and FUEG-Bmi-1 lentivirus used in this study. (B) Immunoblot of whole-cell protein extracts from transduced 293T and GFP+FACS-sorted human CB-derived CD34+ cells with anti-Bmi-1 and anti-Actin antibodies.
Fig. 2
Ectopic Bmi-1 expression in CB CD34+ cells enhances the formation of both primary and secondary erythroid colonies. Numbers of erythroid colonies in primary (A) and secondary (B) The number of colony-forming cells (CFCs) was significantly increased in FUEG-Bmi-1 group (∗P＜0.05, Error bar is not shown). (C) Representative images for bright field (BF) and GFP expression (GFP) of each type of colonies from primary CFC assay in FUEG-Bmi-1 group. (D) Representative images for GFP expression of CFU-E and cytospin images of the cells obtained from secondary colonies in FUEG-Bmi-1 group.
Fig. 3
The increase in number of erythroid colonies in the Bmi-1 CD34+ group is dependent on erythropoietin (Epo). Methylcellulose culture was established without Epo, with 1 unit/ml, and 6 units/ml of Epo, rspectively (A, B, C, respectively). KL (10ng/mL) was added in all cases. Significant differences in the number of erythroid colonies and in the total number of colonies were observed in the presence of 6 IU/ml of Epo (∗P＜0.05).
Fig. 4
Forced expression of Bmi-1 does not increase self-renewal of CB HSC. (A) Results of cobblestone area forming assay showed that there is no significant difference between non-transduced, FUEG and FUEG-Bmi-1-transduced groups (Error bar is not shown). (B, D) Number of non-adherent cells in co-cultures of CD34+ cells on OP-9 and MS-5 stromal cells was not different between the two groups (Error bar is not shown). (C, E) Methylcellulose cultures were established with non-adherent cells obtained from 5-week co-cultures on OP-9 and MS-5 stromal cells. A significant decrease in total number of 5 week colonies in FUEG-Bmi-1 group was observed.
Fig. 5
Overexpression of Bmi-1 promotes erythroid differentiation of CB CD34+ cells. GFP+ CB CD34+ cells were cultured in serum-free medium supplemented with KL, Flt3 ligand, Tpo, and Epo. (A) Representative images of flow cytometric analysis using anti-CD34 and anti-Glycophorin A antibodies after 7 days of culture in serum-free condition. (B) Quantification of CD34+ and Glycophorin A+ cells in stroma free culture for 7 days showed a significant decrease of CD34+ cells and an increase of Glycophorin A+ cells. (C) Total number of the cells in stromal-free culture did not show significant difference (Error bar is not shown).
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