kjh The Korean Journal of Hematology Korean J Hematol 1738-7949 2092-9129 Korean Society of Hematology 10.5045/kjh.2007.42.3.241 kjh-42-241 Original Article Enforced Expression of BMI-1 in Postnatal Human CD34+ Cells Promotes Erythroid Differentiation LeeGabsangD.V.M., Ph.D. 1 KimByung SooM.D. Ph.D. 2 ShiehJae-hung 3 MooreMalcolm AS 4 Craniomaxillofacial Life Science 21, College of Dentistry, Seoul National University, Korea Division of Hematology/Oncology, Korea University Medical Center, Seoul, Korea Cell Biology Program, Memorial Sloan Kettering Cancer Center, Korea Cell Biology Program, Memorial Sloan Kettering Cancer Center, Korea Correspondence to：Gabsang Lee, Ph.D., D.V.M. Craniomaxillofacial Life Science 21, College of Dentistry, Seoul National University 275-1, Yeongeon-dong, Jongno-gu, Seoul 110-749, Korea Tel: ＋82-2-740-8663, Fax: ＋82-2-740-8665 E-mail: csuh@amc.seoul.kr 092007 19092007 423241 249 25072007 30082007 03092007 Copyright © 2007 Korean Society of Hematology 2007 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 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.

 Bmi-1 Erythroid differentiation CD34+ cell REFERENCESLund AH van Lohuizen M Polycomb complexes and silencing mechanisms Curr Opin Cell Biol 200416 23946 Buszczak M Spradling AC Searching chromatin for stem cell identity Cell 2006125 2336 Molofsky AV He S Bydon M Morrison SJ Pardal R Bmi-1 promotes neural stem cell self-renewal and neural development but not mouse growth and survival by repressing the p16Ink4a and p19Arf senescence pathways Genes Dev 200519 14327 Molofsky AV Pardal R Iwashita T Park IK Clarke MF Morrison SJ Bmi-1 dependence distinguishes neural stem cell self-renewal from progenitor proliferation Nature 2003425 9627 Christopher FA Dimos JT Ivanova NB Lowry N Lemischka IR Temple S shRNA Knockdown of Bmi-1 Reveals a Critical Role for p21-Rb Pathway in NSC Self-Renewal during Development Cell Stem Cell 20071 12 Park IK Qian D Kiel M Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells Nature 2003423 3025 Iwama A Oguro H Negishi M Enhanced self-renewal of hematopoietic stem cells mediated by the polycomb gene product Bmi-1 Immunity 200421 84351 Lessard J Sauvageau G Bmi-1 determines the proliferative capacity of normal and leukaemic stem cells Nature 2003423 25560 Lessard J Baban S Sauvageau G Stage-specific expression of polycomb group genes in human bone marrow cells Blood 199891 121624 Hemmati HD Nakano I Lazareff JA Cancerous stem cells can arise from pediatric brain tumors Proc Natl Acad Sci USA 2003100 1517883 Mihic-Probst D Kuster A Kilgus S Consistent expression of the stem cell renewal factor BMI-1 in primary and metastatic melanoma Int J Cancer 2007121 176470 Glinsky GV Berezovska O Glinskii AB Microarray analysis identifies a death-from-cancer signature predicting therapy failure in patients with multiple types of cancer J Clin Invest 2005115 150321 Jacobs JJ Kieboom K Marino S DePinho RA van Lohuizen M The oncogene and Polycomb-group gene bmi-1 regulates cell proliferation and senescence through the ink4a locus Nature 1999397 1648 Jacobs JJ Scheijen B Voncken JW Kieboom K Berns A van Lohuizen M Bmi-1 collaborates with c-Myc in tumorigenesis by inhibiting c-Myc-induced apoptosis via INK4a/ARF Genes Dev 199913 267890 Zhang P Iwasaki-Arai J Iwasaki H Enhancement of hematopoietic stem cell repopulating capacity and self-renewal in the absence of the transcription factor C/EBP alpha Immunity 200421 85363 Oneal PA Gantt NM Schwartz JD Fetal hemoglobin silencing in humans Blood 2006108 20816 Weinberg RA The retinoblastoma protein and cell cycle control Cell 199581 32330 Walkley CR Shea JM Sims NA Purton LE Orkin SH Rb regulates interactions between hematopoietic stem cells and their bone marrow micro-environment Cell 2007129 108195 Figures

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.

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.

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).

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.

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).