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Supplementary Information.
Park et al. Bmi-1 is required for maintenance of adult self-renewing hematopoietic stem cells.
Methods
Mice Bmi-1+/- mice were backcrossed onto a C57BL/Ka-Thy1.1 background at least five times
in the animal facility at the University of Michigan Cancer center. Bmi-1-/- mice were generated
by mating heterozygotes. For timed pregnancy, the presence of a vaginal plug at noon of the next
day was considered as day 0.5 dpc. For most of experiments, 4 to 8 week-old mice were used.
C57Bl/6J and p53 deficient mice in a C57Bl/6J background are purchased from the Jackson
Laboratories (Bar Harbor, Maine).
RT-PCR. For RT-PCR using 10 cells and single cells, total RNA was isolated using the Trizol
Reagent (Invitrogen), and total RNA was used for oligo-dT primed cDNA synthesis in the
presence or absence of SuperscriptII reverse transcriptase. All of the reaction was diluted 10 fold
in the first round PCR and amplified using Advantage polymerase mix (BD Biosciences,
Clontech) in the presence of both gene specific and -actin outer primers. Two l each were used
separately for the secondary PCR for bmi-1 and -actin in a 25-l reaction. PCR conditions were
as follow: 1 cycle of 94 °C for 2 min, 30 to 35 cycles of 94 °C for 20 sec/68 °C for 3 min. For
semi-quantitative PCR, 2 to 5 g of total RNA was used for reverse transcription, and 1/10th of
the reaction was used for PCR. Sequences of PCR primers are listed in Supplemental Table 1.
Chemotaxis of hematopoietic stem cells. The chemotaxis assay using lineage-depleted E14.5
fetal liver cells was performed using 100 ng/ml SDF-1 as described1. Briefly, fetal liver cells
were stained with lineage markers, and the lineage positive cells were removed using anti-rat
IgG magnetic bead (Dynabeads, Dynal A.S., Oslo, Norway). The remaining cells were incubated
for 1 hr at 37 C in RPMI medium with 10 % serum and used for chemotaxis assays using the
transwell as follows. Eight hundred thousand cells in 100-l medium were placed in the top
compartment and 600 l of medium containing 100 ng/ml SDF-1in the bottom chamber of the
transwell (24-well, 5 micron barrier filter, Costar) in 3 to 10 replicates. After 3 hr at 37 C, cells
in the bottom chamber were collected and stained for fetal liver HSCs (Thy1.1loSca-1+ Mac1+Lineage-CD4-) and analyzed by flow cytometry.
Microarray analysis. Total RNA isolated from bone marrow of each of four wild type and
bmi-1-/- mice was used for microarray analysis using Affymetrix Murine Genome Array U74Av2
chips (Afftmetrix, Santa Clara, CA). Probe preparation and hybridization were done at the
UMCCC DNA Microarray Core at the University of Michigan according to the protocol
provided by Affymetrix. Equal amounts of RNA from wild type and bmi-1-/- bone marrow cells
were used for analysis. Although the bmi-1-/- mice had lower numbers of bone marrow
mononuclear cells, the frequency of progenitor cells as measured by flow cytometry (Fig. 1b)
and colony forming cells (Supplemental Fig. 1d) was similar in both groups of mice. The
expression of CD45 was similar in both groups, suggesting that the contribution of hematopoietic
cells to the gene expression analysis was virtually the same in both groups of mice. Expression
values for each gene on each chip were calculated using dChip (www.dChip.org). Differentially
expressed genes were detected using Significance Analysis of Microarrays (SAM), a program
that performs two-sample t-tests based on a permuted deficient distribution with multiplicity
adjustment using false discovery rate (FDR). Genes that were identified as being differentially
expressed were confirmed by RT-PCR.
Figure Legends
Supplemental Fig. 1. Expression of bmi-1 in mouse (a) and human (b) HSCs. Total RNA was
isolated from 1 cell or 10 cell aliquots of adult HSCs (Thy1.1lowc-kit+Sca-1+Lineage- phenotype)
and fetal liver HSCs (Thy1.1lowMac-1+Sca-1+Lineage- phenotype) from C57Bl/Ka-Thy1.1 mice
and used for RT-PCR as described in the Methods. Bmi-1 was detected in all 10 cell aliquots of
both fetal liver and adult bone marrow HSCs, while it was detected in 10 out of 16 single fetal
liver HSCs and 4 out of 15 single cell aliquots of post natal bone marrow HSCs (a). We also
tested
human
normal
(CD34+CD90+CD38-Lineage-)
and
leukemic
stem
cells
(CD34+IL3R+CD38-Lineage-) for bmi-1 expression. Similar to the mouse, bmi-1 was expressed
in both normal HSCs and leukemic stem cells (b). For 10-cell RT-PCR of human HSCs, each
lane represents independently sorted cells.
Supplemental Fig. 2. Analysis of hematopoiesis in bmi-1+/+, bmi-1+/-, and bmi-1-/- mice. a-c,
Peripheral blood from adult bmi-1+/+ (white bars) and bmi-1-/- (black bars) mice was analyzed by
complete blood counts with manual differential. Leukocyte count (a), hematocrit (b), platelet
count (c), and colony forming assays (d) are shown. One thousand bone marrow cells were
plated on methylcellulose containing a cocktail of cytokines. The numbers shown in the bar area
are percent of CFU-GM (white area), BFU-E (gray area), and CFU-GEMM (black area) in the
culture. Statistical analysis was done using the unpaired Student’s t-test, and there were no
significant changes in the number of colonies in each group. Note that bmi-1-/- mice have normal
ranges of red blood cell, neutrophil and monocyte counts; however, lymphocyte counts were
greatly reduced. Also, note that the bmi-1-/- colony size was consistently smaller (Supplemental
Fig. 2d). The above results are in agreement with a previous report that bmi-1 plays a critical role
in B- and T-lymphocyte development2.
Supplemental Figure 3 Total HSC and MPP numbers. Total HSC (a) and MPP (b) numbers
were calculated by multiplying the HSC or MPP frequency and total bone marrow cells present
in the long bones of the mouse.
Supplemental Figure 4 Fetal liver HSC migration. a, Chemotaxis of fetal liver HSCs in
response to SDF-1. Lineage-depleted fetal liver cells were placed on transwell and flow
cytometry was used to determine the number of fetal liver HSCs that migrated in response to
SDF-1. Assays were done in 3 to 10 replicates, and repeated three times. A representative
experiment with 10 replicates is shown. The p values were calculated using the Student t-test. No
significant differences in the number of migrated cells among wild type, heterozygous and
knockout fetal liver HSCs were detected, indicating that fetal liver stem cells from bmi-1-/- and
bmi-1+/+ mice have similar abilities to migrate towards a SDF-1 gradient. b, Expression of
CXCR4 and SDF-1 in fetal liver and bone marrow. RT-PCR was performed as described in the
Methods. At the end of cycle 22, 25, 28 and 31, aliquots were taken and analyzed on an agarose
gel. -actin was used as an internal control. There are no changes in expression of either SDF-1
or CXCR4 in E14.5 fetal liver cells and adult bone marrows from bmi-1-/- mice, consistent with
the chemotaxis results. Although these results suggest the HSC defect in bmi-1-/- bone marrow is
not likely to be the result of defective fetal liver HSC migration to the bone marrow, it is possible
that bmi-1 affects homing via other pathways.
Supplemental Figure 5 Semi-quantitative RT-PCR of candidate genes regulated by bmi-1.
cDNA was reverse transcribed from 2 g total RNA and gene specific primers were used for
PCR of p16Ink4a (a), p19Arf (b), Hoxa9 (c), Hoxb4 (d) and -actin (e). For p16Ink4a, p19Arf and
Hoxb4, aliquots were taken at the end of 26th, 29th, 32nd, and 35th cycle, for Hoxa9, at 21st, 24th,
37th, and 40th cycle, and for -actin, 17th, 20th, 23rd, and 26th cycle. Bmi-1 deletion led to elevated
expression of both p16Ink4a and p19Arf in all tissues except in fetal liver p19Arf expression was not
affected. Hoxa9 expression is higher in early progenitor cells than in HSCs3, which is consistent
with the observation that Hoxa9-/- mice have defects in hematopoietic progenitor cells, but not in
HSCs4. Expression of Hoxa9 was elevated in the thymus and bone marrow of bmi-1-/- mice.
These results were reconfirmed by real time PCR (data not shown). Whether bmi-1 deletion
causes Hoxa9 upregulation in adult HSCs is not possible to test since we were not able to isolate
this population in bmi-1-/- mice. The human HOXB3 mRNA level is high in the early stem,
progenitor cells and declines during development5. Over-expression of HOXB3 in hematopoietic
cells via retroviral gene transfer causes defective lymphoid development and progressive myeloproliferation, suggesting roles for HOXB3 in the proliferation and differentiation of both early
myeloid and lymphoid developmental pathways6. To examine whether mouse Hoxb3 expression
is altered in bmi-1-/- mice, RT-PCR was performed. Hoxb3 was expressed at low levels in all
tissues examined, and there were no significant differences in Hoxb3 levels among wild type,
heterozygote and knockout mice (data not shown). This suggests that bmi-1 is not required for
Hoxb3 expression. HoxB4 is thought to be important for HSC development7,8. Hoxb4 level was
not changed in bmi-1-/- mice, indicating that bmi-1 does not repress this gene. These results
suggest that Bmi-1 does not suppress the expression of Hox members important for HSCs.
Supplemental Table 1. List of oligonucleotides used in RT-PCR.
Supplemental Table 2. Partial list of genes differentially expressed in bmi-1-/- mice. Total RNA
isolated from each of four wild type and bmi-1-/- mice were used for the gene expression analyses
using Affymetrix Murine Genome Array U74Av2 chips. Fold change larger than 1 indicates
increased expression and smaller than 1 decreased expression in bmi-1-/- mice.
Supplemental Table 3. Complete list of genes differentially expressed in bone marrow cells of
bmi-1-/- mice identified by microarray. An explanation of the output is given below.
Row
Gene Name
Gene ID
Score(d)
Numerator(r)
Denominator(s+s0)
Fold
Change
q-value (%)
10359
carbonic anhydrase 3
AJ006474
7.9729918
2.0865575
0.2617032
4.34667
1.2195122
Row number from original data
di  score  
ri  numerator 
si  s0  denominator 
This is equivalent to a t-test
Fold change = KO/WT
q-value is equivalent
to a p-value
Supplemental References
1.
2.
3.
4.
5.
6.
7.
8.
Wright, D. E. et al. Hematopoietic stem cells are uniquely selective in their migratory
reponse to chemokines. J. Exp. Med. 195, 1145-1154 (2002).
van der Lugt, N. M. et al. Posterior transformation, neurological abnormalities, and
severe hematopoietic defects in mice with a targeted deletion of the bmi-1 protooncogene. Genes & Development 8, 757-769 (1994).
Park, I. K. et al. Differential gene expression profiling of adult murine hematopoietic
stem cells. Blood 99, 488-498 (2002).
Lawrence, H. J. et al. Mice bearing a targeted interruption of the homeobox gene
HOXA9 have defects in myeloid, erythroid, and lymphoid hematopoiesis. Blood 89,
1922-1930 (1997).
Sauvageau, G. et al. Differential Expression of Homeobox Genes in Functionally Distinct
CD34+ Subpopulations of Human Bone Marrow Cells. PNAS 91, 12223-12227 (1994).
Sauvageau, G. et al. Overexpression of HOXB3 in hematopoietic cells causes defective
lymphoid development and progressive myeloproliferation. Immunity 6, 13-22 (1997).
Antonchuk, J., Sauvageau, G. & Humphries, R. K. HOXB4 overexpression mediates very
rapid stem cell regeneration and competitive hematopoietic repopulation. Exp. Hematol.
29, 1125-34. (2002).
Buske, C. et al. Deregulated expression of HOXB4 enhances the primitive growth
activity of human hematopoietic cells. Blood 100, 862-868 (2002).