Korean J Hematol.  2007 Sep;42(3):241-249. 10.5045/kjh.2007.42.3.241.

Enforced Expression of BMI-1 in Postnatal Human CD34+ Cells Promotes Erythroid Differentiation

• Lee G
• Kim BS
• Shieh JH
• Moore MA

• KMID: 2305199
• DOI: http://doi.org/10.5045/kjh.2007.42.3.241

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.
CONCLUSION
Our data suggest that ectopic expression of Bmi-1 in human hematopoietic stem/progenitor cells may result in the differentiation to the erythroid lineage rather than promoting self-renewal.

Keyword

Bmi-1; Erythroid differentiation; CD34+ cell

MeSH Terms

Cell Proliferation
Coculture Techniques
Erythropoiesis
Erythropoietin
Fetal Blood
Glycophorin
Hematopoietic Stem Cells
Humans*
Lentivirus
Stem Cells
Erythropoietin
Glycophorin

Figure

• 

  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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