HEMATOPOIESISAND STEM CELLS
Elisa Bianchi,1Roberta Zini,1Simona Salati,1Elena Tenedini,1,2Ruggiero Norfo,1Enrico Tagliafico,1-3Rossella Manfredini,1
and Sergio Ferrari1,3
1Department of Biomedical Sciences, Biological Chemistry Section, University of Modena and Reggio Emilia, Modena, Italy;2BioPharmaNet, Emilia-Romagna
High-Tech Network, Ferrara, Italy; and3Center for Genome Research, University of Modena and Reggio Emilia, Modena, Italy
The c-myb transcription factor is highly
expressed in immature hematopoietic
cells and down-regulated during differen-
tiation. To define its role during the hema-
topoietic lineage commitment, we silenced
progenitor cells. Noteworthy, c-myb silenc-
ing increased the commitment capacity
toward the macrophage and megakaryo-
cyte lineages, whereas erythroid differen-
tiation was impaired, as demonstrated by
clonogenic assay, morphologic and im-
munophenotypic data. Gene expression
profiling and computational analysis of
promoter regions of genes modulated in
transcription factors Kruppel-Like Factor
1 (KLF1) and LIM Domain Only 2 (LMO2)
as putative targets, which can account for
c-myb knockdown effects. Indeed, chro-
reporter assay demonstrated that c-myb
binds to KLF1 and LMO2 promoters and
transactivates their expression. Consis-
expression of either KLF1 or LMO2 par-
tially rescued the defect in erythropoiesis
caused by c-myb silencing, whereas only
KLF1 was also able to repress the
megakaryocyte differentiation enhanced
in Myb-silenced CD34?cells. Our data
a pivotal role in human primary hemato-
mitment, by enhancing erythropoiesis at
the expense of megakaryocyte diffentia-
transactivation as the molecular mecha-
sus megakaryocyte cell fate decision.
Hematopoiesis is a tightly regulated process involving the ordered
regulation of gene expression throughout development, from the
hematopoietic stem cells (HSCs) to mature and fully differentiated
cells. The c-myb gene encodes for a basic helic turn helix
transcription factor composed of 3 functional domains: a DNA
binding domain at the N terminus, a central transactivation domain
and a C-terminal negative regulatory domain.1
Most of what is known about c-myb involves its role in
hematopoiesis, during which it is highly expressed in immature,
proliferating cells of all hematopoietic lineages and down-
regulated during terminal differentiation. The critical role of c-myb
in regulating normal human hematopoiesis was first related to
cell-cycle control, because its down-regulation by antisense oligo-
nucleotides determines a G1phase cell-cycle arrest.2-5However,
several works indicate multiple cellular roles for this transcription
factor during normal hematopoiesis, showing that c-myb targets
some differentiation-related genes, such as CD34,6c-kit,7c-myc,8
GATA1,9neutrophil elastase,10and myeloperoxidase.11
c-myb is essential for the hematopoietic system development,
because c-myb?/?mice die at E15 due to failure of fetal hepatic
erythropoiesis.12c-myb knockout and knockdown models collec-
tively demonstrate that c-myb plays a pivotal role at multiple stages
of hematopoiesis,12,13but the c-myb target genes responsible for
these effects remain largely unknown. Moreover, little is known
about the role of c-myb in human adult hematopoietic differentia-
tion and in particular on the molecular mechanisms governing
To investigate the role of c-myb in the human hematopoietic
commitment and to get new insights into the molecular mecha-
nisms underlying c-myb–driven lineage specification, we silenced
c-myb in human CD34?stem/progenitor cells and primary myelo-
blasts in vitro. Our data showed that c-myb silencing in CD34?
cells forces their commitment toward the mono-macrophage and
megakaryocyte lineages whereas the erythroid and granulocyte
ones are strongly impaired. Moreover, microarrays data, together
with chromatin immunoprecipitation (ChIP) and Luciferase assays
expression as a new molecular mechanism through which c-myb
Human CD34?stem/progenitor cells purification
Human CD34?cells were purified from umbilical cord blood (CB)
samples, collected after normal deliveries, according to the institutional
guidelines for discarded material. Mononuclear cells were isolated by
Ficoll-Hypaque (Lymphoprep; Nycomed Pharma) gradient separation,
washed twice with phosphate-buffered saline, and then CD34?cells were
Submitted August 13, 2009; accepted June 28, 2010. Prepublished online as
Blood First Edition paper,August 4, 2010; DOI 10.1182/blood-2009-08-238311.
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
© 2010 by TheAmerican Society of Hematology
e99 BLOOD, 25 NOVEMBER 2010?VOLUME 116, NUMBER 22
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purified by immunomagnetic sorting (EasySep Human CD34 Positive
Selection kit, StemCell Technologies Inc.). The purity of CD34?cells,
assessed by flow cytometry, was always ? 95% (data not shown). After
immunomagnetic separation, CD34?cells were seeded in 24-well plates at
5 ? 105/mL in Iscove modified Dulbecco medium (IMDM; GIBCO)
containing 20% Human Serum (Bio-Whittaker), stem cell factor (SCF;
50 ng/mL), Fms-like tyrosine kinase 3 ligand (Flt3L; 50 ng/mL), TPO
(thrombopoietin; 20 ng/mL), interleukin-6 (IL-6; 10 ng/mL), and IL-3
(10 ng/mL; all from R&D Systems), and electroporated 24 hours later.
Human CD14?myeloblasts purification
Human CD14?myeloblasts were obtained by CB CD34?stem/progenitor
cells liquid culture according to Montanari M et al14and purified by
immunomagnetic sorting (EasySep Human CD14 Positive Selection kit;
StemCell Technologies Inc). The detailed procedure of CD14?cells
purification is reported in supplemental Methods (available on the Blood
Web site; see the Supplemental Materials link at the top of the online
article).The purity of CD14?cells, assessed by flow cytometry, was always
? 95% (data not shown).
After immunomagnetic separation, CD14?cells were seeded in 6-well
plates at 106/mL in IMDM, added with 20% fetal calf serum (Bio-
Whittaker), in the presence of human hematopoietic cytokines, namely SCF
(10 ng/mL), Flt3L (10 ng/mL), IL-6 (10 ng/mL), and IL-3 (10 ng/mL; all
from R&D Systems) and electroporated 24 hours later.
The electroporation of CD34?stem/progenitor cells and CD14?myelo-
blasts was performed using the Amaxa Nucleofector® Technology as
A mix of 3 Silencer Pre-designed siRNAs targeting human c-myb
(supplemental Table 1) was used (Ambion).
Each sample was electroporated 3 times, once every 24 hours. For each
electroporation, 5 ? 105CD34?cells or 107CD14?cells were resuspended
in 100 ?L of Human CD34?Nucleofection Solution (Amaxa Biosystem),
containing 5 ?g of siRNAand pulsed with the program U-01 (CD34?cells)
or X-01 (CD14?cells). Transfection efficiency was evaluated by the
nucleofection of a nontargeting Alexa Fluor 488–conjugated siRNA
(QIAGEN) followed by flow cytometric analysis at 6 hours after transfec-
tion. To exclude nonspecific effects of siRNA nucleofection, for each
experiment, one sample electroporated without siRNAs (MOCK) and one
transfected with a nontargeting siRNA (NegCTR; siCONTROL Non-
Targeting Pool, Dharmacon) were performed.
After each transfection, CD34?cells were transferred into prewarmed fresh
medium in 24-well plates and maintained in the same culture conditions as
before (see “Human CD34?stem/progenitor cells purification”). Cells were
analyzed 24 and 48 hours after the last nucleofection (also reported as
“postnucleofection,” meaning 4 and 5 days after cell purification, respec-
tively) for both cell viability and c-myb expression. For liquid culture
differentiation assays, the 24-hour postnucleofection CD34?cells were
plated (5 ? 105/mL) in IMDM added with 20% BIT 9500 serum substitute
(bovine serum albumin, insulin, and transferrin; StemCellTechnologies), in
order to set up erythroid (erythropoietin, EPO, 0.4 U/mL, SCF 50 ng/mL),
megakaryocyte (TPO, 100 ng/mL),16granulocyte colony stimulating factor
(GCSF, 25 ng/mL), and monocyte (macrophage colony-stimulating factor,
100 ng/mL; SCF, 20 ng/mL; IL6, 20 ng/mL; and Flt3L, 50 ng/mL)17
unilineage cultures in addition to multilineage cell cultures (SCF, 50 ng/
mL; Flt3L, 50 ng/mL;TPO, 20 ng/mL; IL-3, 10 ng/mL; IL-6, 10 ng/mL; all
cytokines from R&D Systems). The medium was replaced every 3 days,
whereas for granulocyte unilineage culture GCSF 25 ng/mL was added
everyday. Differentiation was assessed by morphological analysis of
May-Grunwald-Giemsa–stained cytospins and by flow cytometric analysis
of CD14, CD15, CD163, Glycophorin A (GPA), and CD41 antigen
expression at 5, 7, 9, 11, and 13 days after nucleofection (ie, 8, 10, 12, 14,
and 16 days after CD34?cell purification), respectively.
CD14?myeloblasts culture conditions
After each transfection, myeloblasts were seeded in 6-well plates (106/mL)
into prewarmed IMDM, added with 20% fetal calf serum and human
cytokines, namely SCF (10 ng/mL), Flt3L (10 ng/mL), IL-6 (10 ng/mL),
and IL-3 (10 ng/mL). Cells were analyzed 24 and 48 hours after nucleofec-
tion for both cell viability and c-myb expression. Twenty-four hours after
nucleofection, myeloblasts were seeded in the same culture conditions as
described above and treated with all-trans retinoic acid (ATRA) 10?6M
(Sigma-Aldrich) or GCSF 25 ng/mL (added everyday) for 3 and 10 days,
respectively (ie, up to days 4 and 11 after nucleofection).
Differentiation was assessed by flow cytometric analysis of CD14
antigen expression 4 days after nucleofection and by morphological
analysis of May-Grunwald-Giemsa–stained cytospins at day 4 after nucleo-
fection for both untreated and ATRA-treated cells and at day 11 after
nucleofection for GCSF-treated cells.
of MOCK, NegCTR and siRNA-treated (MYBsiRNA) cells, obtained from 3
independent experiments, were converted in biotinilated cRNAaccording to the
Human HG-U133AGeneChip arrays hybridization, staining, and scanning were
The GeneChip Operating Software (GCOS) absolute analysis algorithm
was used to determine the amount of a transcript mRNA (signal), whereas
the GCOS comparison analysis algorithm was used to compare gene
expression levels between 2 samples.
Present genes were selected as the sequences showing the Detection call
“P” and Signal ? 100 at least in one sample. Differentially expressed genes
were selected as the sequences showing a Change call “I” or “D” and Signal
Log Ratio (SLR)??1 or??1 (Fold Change?2SLR) in both the pairwise
DAVID TOOL 2008 (http://david.abcc.ncifcrf.gov/) software was used
to examine selected lists of genes to identify overrepresentation of
functional classes accordingly with gene ontology (GO) classification. All
of the data have been deposited in the Gene Expression Omnibus
MIAME-compliant public database (National Center for Biotechnology
for CD34?cells and http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc?
GSE21943 for CD14?myeloblasts.
The methods for CD14?myeloblasts purification, Western blot detec-
tion of c-myb, KLF1, and LMO2 protein levels, methylcellulose and
collagen clonogenic assays, morphological and immunophenotypic analy-
sis, RNA extraction; quantitative real-time polymerase chain reaction
(qRT-PCR), Myb kinetics during erythroid differentiation, ChIP; luciferase
reporter assay; retroviral vectors construction and packaging, and CD34?
cells transduction, purification, and nucleofection are described in supple-
The statistics used for data analysis was based on the 2-tail Student t test for
averages comparison in paired samples. In silencing experiments, MOCK
vs NegCTR, MOCK vs. MYBsiRNA, and NegCTR vs MYBsiRNA were
compared. Data were analyzed by Microsoft Excel Software (Version
2007), and P ? .05 was considered significant.
c-myb silencing in CD34?stem/progenitor cells
To get further insights into the role of c-myb in regulating
human primary CB CD34?hematopoietic stem/progenitor cells
using a siRNA approach. We applied the Nucleofector technology
(Amaxa) by optimizing CD34?cells nucleofection protocol as
e100BIANCHI et al BLOOD, 25 NOVEMBER 2010?VOLUME 116, NUMBER 22
For personal use only. on November 5, 2015. by guest
2006 (contract no. 2006057308), Regione Emilia Romagna (area
1b, “Medicina Rigenerativa”) and by Associazione Italiana per la
Ricerca sul Cancro (AIRC) 2007. E.B. is the recipient of a
fellowship from AIL (Italian Association Against Leukemia). S.S.
is the recipient of a fellowship from FIRC (Italian Foundation for
Cancer Research). E.T. is a BioPharmaNet fellow.
biological effects characterization; R.Z. performed microarray
analysis and luciferase assays; S.S. performed chromatin immuno-
precipitation; E. Tenedini performed microarray analysis; R.N.
performed luciferase assays; E. Tagliafico performed computa-
tional analysis of putative c-myb targets; and R.M. and S.F.
designed research and wrote the paper.
Conflict-of-interest disclosure: The authors declare no compet-
ing financial interests.
Correspondence: Rossella Manfredini, Department of Biomedi-
cal Sciences, Biological Chemistry Section, University of Modena
and Reggio Emilia, via Campi 287, 41100 Modena, Italy; e-mail:
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e110BIANCHI et alBLOOD, 25 NOVEMBER 2010?VOLUME 116, NUMBER 22
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online August 4, 2010
2010 116: e99-e110
Rossella Manfredini and Sergio Ferrari
Elisa Bianchi, Roberta Zini, Simona Salati, Elena Tenedini, Ruggiero Norfo, Enrico Tagliafico,
c-myb supports erythropoiesis through the transactivation of KLF1 and
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