1. Repression of BMI1 in normal and leukemic human CD34+ cells impairs self-renewal and induces apoptosis
by Aleksandra Rizo, Sandra Olthof, Lina Han, Edo Vellenga, Gerald de Haan, and Jan Jacob Schuringa
Blood
Volume 114(8):
August 20, 2009
©2009 by American Society of Hematology

2. Down-modulation of BMI1 by lentiviral RNAi in human CB CD34+ cells impairs proliferation and reduces CFC and LTC-IC frequencies.
Down-modulation of BMI1 by lentiviral RNAi in human CB CD34+ cells impairs proliferation and reduces CFC and LTC-IC frequencies. (A) CB CD34+ cells were transduced with control (scrambled) scr-RNAi or BMI1-RNAi particles and sorted; mRNA was isolated; and BMI1 expression was analyzed by quantitative reverse transcription (RT)–PCR analysis. (B) As in panel A, but now total lysates were prepared and analyzed by Western blotting. (C) Transduced CB cells were grown in long-term cocultures on MS5 bone marrow stromal cells. Cultures were weekly analyzed, and the growth curve represents cumulative cell numbers during the culture period. A representative experiment of 3 independent experiments is shown. (D) Hematopoietic differentiation was analyzed by FACS on suspension cells from MS5 cocultures at week 1. (E) Suspension cells were harvested from MS5 cocultures, as described in panel C, and progenitor content was determined by CFC assays in methylcellulose. (F-G) Lentiviral transductions as in panel A, but now stem cell frequencies were determined in limiting dilution (F) or in bulk T25 flasks (G).
Aleksandra Rizo et al. Blood 2009;114:
©2009 by American Society of Hematology

3. Knockdown of BMI1 reduces cell growth and CFC formation in liquid culture conditions.
Knockdown of BMI1 reduces cell growth and CFC formation in liquid culture conditions. (A) CB CD34+ cells were transduced with control (scrambled) scr-RNAi or BMI1-RNAi particles, sorted, and plated in stroma-free liquid conditions (IMDM supplemented with 10% FCS, 10 ng/mL IL-3, and 100 ng/mL TPO). Cumulative expansion is shown of a representative experiment of 3 independent experiments. (B) Progenitor content was determined by CFC assays in methylcellulose at week 2 of liquid culture. CFCs per 10 000 cells (left panel) as well as the total amount of generated CFCs (right panel) are shown. (C) Representative micrographs of colonies in methylcellulose displaying a reduction in colony size after BMI1 knockdown in cells from week 2 liquid cultures. (D) Hematopoietic differentiation was analyzed by FACS on suspension cells from MS5 cocultures at week 1.
Aleksandra Rizo et al. Blood 2009;114:
©2009 by American Society of Hematology

4. BMI1 knockdown induces apoptosis in CD34+38− cells.
BMI1 knockdown induces apoptosis in CD34+38− cells. CB CD34+ cells were transduced with control (scrambled) scr-RNAi or BMI1-RNAi particles and sorted, and single cells were deposited in 96-well plates and cultured in stroma-free conditions (IMDM supplemented with 10% FCS, 10 ng/mL IL-3, and 100 ng/mL TPO). Wells were evaluated microscopically 1 day and 5 days after plating, and wells were classified as quiescence if 1 live cell was observed; if multiple cells were observed, they were classified as proliferation; and if no cells were seen, they were classified as apoptosis. A representative experiment of 3 independent experiments is shown, whereby individual 96 wells per group were analyzed.
Aleksandra Rizo et al. Blood 2009;114:
©2009 by American Society of Hematology

5. Apoptosis induced by BMI1 down-modulation coincides with elevated ROS accumulation and reduced FOXO3A expression.
Apoptosis induced by BMI1 down-modulation coincides with elevated ROS accumulation and reduced FOXO3A expression. (A) Transduced CB CD34+ cells were cultured in serum-free conditions (HPGM supplemented with SCF and Flt3L) for 10 days, and expansion was monitored. (B) The percentage of apoptotic cells at day 10 was determined by FACS staining for annexin V and PI. (C) Transduced cells were cultured in conditions described in (A), and at day 10 were stained with H2DCFDA to determine the intracellular levels of ROS by FACS. (D) Transduced cells were cultured in the absence or presence of 100 μM NAC for 9 days, after which ROS accumulation was determined by FACS. (E) CFC assays were performed with transduced cells in methylcellulose cultures in the absence or presence of 100 μM NAC. (F) CB CD34+ cells were transduced with control (scrambled) scr-RNAi or BMI1-RNAi, and CD34+38− GFP+ cells were sorted on glass slides. Immunohistochemical staining was performed using antibodies against FOXO3A.
Aleksandra Rizo et al. Blood 2009;114:
©2009 by American Society of Hematology

6. BMI1 is required for long-term growth and self-renewal of AML CD34+ cells.
BMI1 is required for long-term growth and self-renewal of AML CD34+ cells. (A) AML CD34+ cells from different FAB subclassification were transduced with scrambled RNAi or BMI1-RNAi lentiviral vectors, and long-term cultures on MS5 bone marrow stromal cells were performed. Expansion was monitored weekly, and cumulative cell counts are shown. (B) Experiment as in panel A, but now transduced AML CD34+ cells were cultured on MS5 for a period of 5 to 6 weeks, after which human CD45+ cells were harvested and replated onto new MS5 cells, followed by an additional culturing period of 4 to 5 weeks. (C) Representative micrographs of cobblestone area-forming cells present in MS5 cocultures at week 4 initiated with AML CD34+ cells transduced with scrambled RNAi or BMI1-RNAi lentiviral vectors. Pictures were taken with a Leica DM-IL microscope (Leica Microsystems) with a 20×/0.30 objective.
Aleksandra Rizo et al. Blood 2009;114:
©2009 by American Society of Hematology

7. Down-regulation of BMI1 expression in CB or AML CD34+ cells results in derepression of p14ARF and p16INK4a.
Down-regulation of BMI1 expression in CB or AML CD34+ cells results in derepression of p14ARF and p16INK4a. CB or AML CD34+ cells were transduced with scrambled RNAi or BMI1-RNAi lentiviral vectors and sorted, and RNA was isolated. Quantitative RT-PCRs were performed to determine the expression levels of BMI1, p14ARF, and p16INK4a. As a control, BMI1 was overexpressed in CB CD34+ cells, and quantitative RT-PCR analysis was performed (right panels).
Aleksandra Rizo et al. Blood 2009;114:
©2009 by American Society of Hematology