Fancd2-/- mice have hematopoietic defects that can be partially corrected by resveratrol.
ABSTRACT Progressive bone marrow failure is a major cause of morbidity and mortality in human Fanconi Anemia patients. In an effort to develop a Fanconi Anemia murine model to study bone marrow failure, we found that Fancd2(-/-) mice have readily measurable hematopoietic defects. Fancd2 deficiency was associated with a significant decline in the size of the c-Kit(+)Sca-1(+)Lineage(-) (KSL) pool and reduced stem cell repopulation and spleen colony-forming capacity. Fancd2(-/-) KSL cells showed an abnormal cell cycle status and loss of quiescence. In addition, the supportive function of the marrow microenvironment was compromised in Fancd2(-/-) mice. Treatment with Sirt1-mimetic and the antioxidant drug, resveratrol, maintained Fancd2(-/-) KSL cells in quiescence, improved the marrow microenvironment, partially corrected the abnormal cell cycle status, and significantly improved the spleen colony-forming capacity of Fancd2(-/-) bone marrow cells. We conclude that Fancd2(-/-) mice have readily quantifiable hematopoietic defects, and that this model is well suited for pharmacologic screening studies.
- SourceAvailable from: Giovanni PaganoFree Radical Biology and Medicine. 01/2013; 58:118-125.
- [Show abstract] [Hide abstract]
ABSTRACT: Fanconi anaemia (FA) is a recessive disorder characterized by genomic instability, congenital abnormalities, cancer predisposition and bone marrow (BM) failure. However, the pathogenesis of FA is not fully understood partly due to the limitations of current disease models. Here, we derive integration free-induced pluripotent stem cells (iPSCs) from an FA patient without genetic complementation and report in situ gene correction in FA-iPSCs as well as the generation of isogenic FANCA-deficient human embryonic stem cell (ESC) lines. FA cellular phenotypes are recapitulated in iPSCs/ESCs and their adult stem/progenitor cell derivatives. By using isogenic pathogenic mutation-free controls as well as cellular and genomic tools, our model serves to facilitate the discovery of novel disease features. We validate our model as a drug-screening platform by identifying several compounds that improve hematopoietic differentiation of FA-iPSCs. These compounds are also able to rescue the hematopoietic phenotype of FA patient BM cells.Nature Communications 07/2014; 5. · 10.74 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Androgens are widely used for treating Fanconi anemia (FA) and other human bone marrow failure syndromes, but their mode of action remains incompletely understood. Aged Fancd2(-/-) mice were used to assess the therapeutic efficacy of oxymetholone (OXM) and its mechanism of action. Eighteen-month-old Fancd2(-/-) mice recapitulated key human FA phenotypes, including reduced bone marrow cellularity, red cell macrocytosis, and peripheral pancytopenia. As in humans, chronic OXM treatment significantly improved these hematological parameters and stimulated the proliferation of hematopoietic stem and progenitor cells. RNA-Seq analysis implicated downregulation of osteopontin as an important potential mechanism for the drug's action. Consistent with the increased stem cell proliferation, competitive repopulation assays demonstrated that chronic OXM therapy eventually resulted in stem cell exhaustion. These results expand our knowledge of the regulation of hematopoietic stem cell proliferation and have direct clinical implications for the treatment of bone marrow failure. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.Stem cell reports. 11/2014; 4(1).
HEMATOPOIESISAND STEM CELLS
Qing-Shuo Zhang,1Laura Marquez-Loza,1Laura Eaton,1Andrew W. Duncan,1Devorah C. Goldman,1,2PraveenAnur,2,3
Kevin Watanabe-Smith,1R. Keaney Rathbun,2,3William H. Fleming,1,4Grover C. Bagby,2,3and Markus Grompe1
1Oregon Stem Cell Center, Department of Pediatrics, Oregon Health & Science University, Portland, OR;2Knight Cancer Institute, Oregon Health & Science
University, Portland, OR;3Northwest VACancer Research Center, VAMedical Center Portland, Portland, OR; and4Division of Hematology and Medical
Oncology, Department of Medicine, Oregon Health & Science University, Portland, OR
Progressive bone marrow failure is a ma-
jor cause of morbidity and mortality in
human Fanconi Anemia patients. In an
effort to develop a Fanconi Anemia mu-
rine model to study bone marrow failure,
Fancd2 deficiency was associated with a
significant decline in the size of the
c-Kit?Sca-1?Lineage?(KSL) pool and re-
duced stem cell repopulation and spleen
colony-forming capacity. Fancd2?/?KSL
cells showed an abnormal cell cycle sta-
tus and loss of quiescence. In addition,
the supportive function of the marrow
microenvironment was compromised in
Fancd2?/?mice. Treatment with Sirt1-
trol, maintained Fancd2?/?KSL cells in
quiescence, improved the marrow micro-
environment, partially corrected the ab-
normal cell cycle status, and significantly
improved the spleen colony-forming ca-
pacity of Fancd2?/?bone marrow cells.
We conclude that Fancd2?/?mice have
readily quantifiable hematopoietic de-
fects, and that this model is well suited
for pharmacologic screening studies.
Fanconi anemia (FA) is a rare, autosomal, recessive genetic disorder
associated with severe birth defects, cancer predisposition, and bone
marrow failure. Thirteen causative genes (FANCA, FANCB, FANCC,
PALB2) have been identified and cloned to date, and the encoded
proteins are believed to work together in a common DNA damage-
from DNA damage induced by cross-linking agents.1,2Although defi-
ciency in DNA cross-link repair renders all FA cells susceptible to
cross-linking agents, bone marrow is the most affected organ system.
Mutations in any of the different FA genes almost universally lead to
The pathogenesis of bone marrow failure in FAremains elusive.
Mutations in several genes involved in DNA damage repair,
including Atr, XPD, and Ercc1, caused either hematopoietic stem
cell (HSC) loss or impaired HSC function under conditions of
stress.4-6These studies suggest that the maintenance of genome
integrity is critical for HSC survival and function. However, the
extent to which genotoxicity, resulting from impaired DNAdamage
repair, contributes to bone marrow failure in FA is unclear.7Other
pathways associated with hematopoietic failure, such as altered
cytokine signaling, may also contribute to FA pathogenesis.8,9For
example, levels of proapoptotic cytokines tumor necrosis factor-?
(TNF-?) and interferon-? (IFN-?) are elevated in FAlymphocytes,
bone marrow cells, and FA patient serum samples.10-12FA bone
marrow cells (at least of the C complementation group) are also
hypersensitive to these cytokines and undergo apoptosis when
exposed to even low levels of them.13-15
To better understand FA, multiple murine knockout models, includ-
ing Fanca?/?, Fancc?/?, Fancg?/?, Fancd2?/?, Fanca?/?-Fancc?/?
double, and Fancl?/?mice, have been developed.16Notably, in
contrast to humans with mutations of these genes, anemia has not
been reported in any of these mice, although the bone marrow cells
from the well-characterized Fancc?/?mice have shown reduced
repopulating ability in transplantation experiments.17-20
Similar to human FA patients, Fancd2?/?mice have a higher
incidence of tumors, a phenotype that occurs rarely in Fancc?/?or
other models with a deficient FAcore-complex gene.16,21However,
the hematopoietic properties of Fancd2?/?mice have not been
fully characterized previously. FANCD2 is thought to play a central
role in the FA/BRCA pathway and is well conserved among
different multicellular eukaryotic species, whereas the majority of
FA core complex genes do not exist in many lower eukaryotes.22,23
Interestingly, there is some evidence that patients with FANCD2
mutations have earlier onset and more rapid progression of
hematologic manifestations.24It is also noteworthy that all the
FANCD2 mutations identified in human patients are hypomorphic,
whereas Fancd2?/?mice have null mutations.21,24We therefore
poietic defects. In this study, we characterized hematopoietic
properties of Fancd2?/?mice in detail. Our results show that
Fancd2?/?mice had multiple hematopoietic defects, including
HSC and progenitor loss in early development, abnormal cell-cycle
status and loss of quiescence in hematopoietic stem and progenitor
cells, and compromised functional capacity of HSCs. These readily
quantifiable hematopoietic defects make Fancd2?/?mice well
suited for pharmacologic studies for bone marrow failure. Indeed,
Submitted April 2, 2010; accepted August 18, 2010. Prepublished online as
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
5140BLOOD, 9 DECEMBER 2010?VOLUME 116, NUMBER 24
we conducted studies designed to evaluate the effect of resveratrol
using this murine model.
Resveratrol is a polyphenol found in grapes and red wine.
Although the full spectrum of bioactivity of resveratrol is not
known, it has antioxidant properties and is a known activator of
SirT1 deacetylase. SirT1, the mammalian ortholog of Sir2, is a
member of the Sirtuins family of nicotine adenine dinucleotide–
dependent protein deacetylases involved in the regulation of many
nuclear processes, including genome stability maintenance, cellu-
lar senescence, apoptosis, aging, and longevity.25,26It has been
reported that resveratrol extends yeast lifespan and suppresses
tumorigenesis in mice.27,28More recently, SirT1 has been shown to
be directly involved in DNA double-strand break repair after
oxidative damage and to protect against genome instability.29,30
Therefore, resveratrol treatment could activate SirT1 and poten-
tially benefit FApatients in their battle against oxidative genotoxic-
FApatients, has been previously used in rodents, and is nontoxic,31we
administered resveratrol to Fancd2?/?mice, and found that this agent
All Fancd2 or Fancc mutant mice and ROSA26 transgenic mice were
maintained on the 129S4 background. Heterozygotes were inbred to
generate mutant mice and littermate controls.The resveratrol diet was made
by mixing powdered resveratrol (3,5,4?-trihydroxystilbene; Orchid Chemi-
cals and Pharmaceuticals, Ltd) with common rodent diet (Bio-Serv) and
given to the mice at 250 mg/kg body weight/d. All animals were treated in
accordance with the guidelines of the Institutional Animals Care and Use
Committee. Unless specified otherwise, the mice used were between 3 and
8 months of age.
Bone marrow cells were isolated from femura of either Fancd2 mutant mice
or wild-type controls and treated with 1? red blood cell Lysis Buffer
(eBioscience) to lyse red blood cells. To count total nucleated cells, a small
aliquot of cell suspension was diluted to 1/100 in 3% acetic acid and
counted using a hemocytometer. All the antibodies were obtained from
eBioscience unless otherwise indicated.
For c-Kit?Sca-1?Lineage?(KSL) cells, cells were stained with a KSL
staining cocktail of phycoerythrin (PE)-conjugated anti-mouse lineage
markers (CD3e, CD4, CD5, CD8a, B220, Ter119, NK1.1, Mac1, and Gr1),
conjugated anti-Ly-6A/E (Sca-1) in phosphate-buffered saline (PBS),
supplemented with 1% bovine serum albumin (BSA). Samples were
examined on a Cytopeia Influx cell sorter, and cytometric data were
analyzed using FlowJo software Version 6.4.7 (TreeStar).
Myeloid progenitors and common lymphoid progenitors were analyzed
on an LSR II flow cytometer (BD Biosciences) as previously described.32
For the analysis of myeloid progenitors, bone marrow cells were labeled
with a PE-conjugated lineage mixture (CD3e, CD4, CD8a, B220, Ter119,
Gr1, CD19, and immunoglobulin M) and anti-interleukin-7 receptor-alpha
(IL-7R-?)–PE, anti-Sca-1–PE, anti-c-Kit–APC, anti-CD34–fluorescein iso-
thiocyanate (FITC), and anti-Fc-?RII/III–PE-Cy7 antibodies. Common
myeloid progenitors (CMPs) were defined as IL-7R-??Lin?Sca-1?c-
were defined as IL-7R-??Lin?Sca-1?c-Kit?CD34?Fc-?RII/IIIhigh. For
common lymphoid progenitors (CLPs), cells were stained with a PE-
conjugated lineage mixture (CD3e, CD4, CD8a, B220, Ter119, Gr1, and
Mac1) and anti-IL-7R-?–FITC, anti-Sca-1–PE-Cy7, and anti-c-Kit–APC
antibodies. CLPs were defined as IL-7R-??Lin?Sca-1lowc-Kitlow.
Cell cycle analysis was done as previously described by Wilson et al.33
Briefly, mouse bone marrow cells were stained with a KSL staining
cocktail, fixed in PBS with 2% paraformaldehyde, and permeabilized in
PBS/3% bovine calf serum (BCS) with 0.5% saponin. Cells were then
stained with anti-Ki-67–FITC (BD Biosciences) in PBS/3% BCS, supple-
mented with 10 ?g/mL Hoeschst 33342 (Sigma-Aldrich). Mouse IgG1-
FITC (BD Biosciences) staining was performed in parallel to serve as an
isotype control. For cell cycle analysis on CD34?KSLcells, anti-CD34–PE
was added to the KSLstaining cocktail.
For the measurement of reactive oxygen species (ROS) in KSL cells,
7000 double-sorted KSL cells were stained with 10?M 5-(and-6)-carboxy-
2?,7?-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA; Invitro-
gen) for 30 minutes, followed by flow cytometric analysis.
Cobblestone area–forming cell (CAFC) assay
Murine stromal layers were established on 96-well plates by culturing
freshly isolated bone marrow cells at 2 ? 106cells/mL in MyeloCult
medium (StemCell Technologies), supplemented with 10?6M freshly
prepared hydrocortisone. A stromal layer was established after 2 weeks of
culture at 33°C, with weekly half-medium changes. Hematopoietic progeni-
tor cells within the stromal layer were then inactivated by irradiation
(1500 rad) from a137Cs ?–irradiation source. Seven days later, whole
marrow cells were cultured on irradiated stromal layers at 3 different
densities (3 ? 104, 1.5 ? 104, and 7.5 ? 103cells per well), with 16 wells
for each density. For the assessment of cobblestone colonies, all wells were
examined under a microscope for the presence of 6 or more closely
associated cells underneath the stromal layer. Each well was scored as
positive (? 1 colonies) or negative (no colonies). Colony frequency was
calculated based on Poisson distribution using L-Calc software (StemCell
Technologies). In the case of resveratrol treatment, resveratrol was added
into the freshly prepared medium at a final concentration of 10 or 25?M.
In vivo competitive repopulation assay
Competitive repopulation assays were done as previously described.20All
the mice used were 8-10 weeks old. Briefly, donor bone marrow cells were
isolated from FA mice (Fancc?/?or Fancd2?/?) and ROSA26Tg/Omice. In
the case of Fancd2?/?donors, Fancd2?/?bone marrow cells were mixed
with ROSA26Tg/Obone marrow cells at a 1:1 ratio and then transplanted into
lethally irradiated Fancc?/?recipient mice. In the case of Fancc?/?donors,
Fancc?/?bone marrow cells were mixed with ROSA26Tg/Obone marrow
cells at a 1:1 ratio and then transplanted into lethally irradiated Fancd2?/?
recipient mice. All recipient mice were lethally irradiated at a split dose of
1200 rad (600 rad each, 4 hours apart) 1 day before transplantation.
Nucleated whole bone marrow cells were counted, and 2 million mixed FA
donor cells and ROSA26Tg/Odonor cells were transplanted into each
recipient FA mouse. DNA was isolated from peripheral blood 7-9 months
posttransplantation, and quantitative real-time polymerase chain reaction
(qPCR) analysis was performed to analyze the contribution of FA donor
cells or ROSA26Tg/Odonor cells to nucleated mature blood cells in recipient
mice. The following primers were used for the qPCR amplification:
ROSA26 transgenic allele:
MG1717, 5? CATCAGCCGCTACAGTCAACAG3?;
MG1718, 5? CAGCCATGTGCCTTCTTCCGC3?.
Fancc mutant allele:
MG1711, 5? GAGCTGCCTGATACGGATGCTG 3?;
MG1791, 5? GGGCTGCTAAAGCGCATGCTC 3?.
Fancd2 mutant allele:
MG2279, 5? TGGAAGAGATGAAGGTTACGATTG 3?;
MG2280, 5? GGTTCTATACTGTTGACCCAATGC 3?.
Colony-forming unit-spleen (CFU-S) assay
Recipient mice (wild-type, 8-12 weeks old) were irradiated with a split dose
of 1100 rad (550 rad each, 4 hours apart) 1 day before transplantation. Forty
thousand bone marrow cells from each donor mouse were transplanted into
each recipient mouse. Twelve days posttransplantation, spleens were
harvested and fixed with Bouin fixative solution.
Fancd2 MAINTAINS QUIESCENCEAND MARROW ENVIRONMENT 5141 BLOOD, 9 DECEMBER 2010?VOLUME 116, NUMBER 24
TNF-? levels were measured with enzyme-linked immunosorbent assay
(ELISA) kits (R&D Systems) as previously described.34Fancc shRNA-
treated or nontargeted control shRNA-treated THP1 cells were stably
integrated with the shRNA-expressing cassette. R848 (imidazoquinoline
resiquimod; AXXORA) was used to induce TNF-? production. Cells were
incubated with R848 for 24 hours before resveratrol was added. Resveratrol
treatment lasted 12 hours, and then TNF-? levels were measured.
Unless specified otherwise, P values were calculated by the 2-tailed,
unpaired Student t test using Prism 4.0 software (GraphPad Software Inc).
AP value less than .05 was considered significant.
Fancd2 deficiency caused loss of hematopoietic stem and
To determine whether Fancd2?/?mice have hematopoietic defects,
we first performed a complete blood count (CBC) on blood samples
from 5-6-month-old Fancd2?/?or Fancd2?/?mice. Despite a
significant drop in platelet numbers in the blood of Fancd2?/?mice
(P ? .05), all the other hematologic parameters measured for both
genotypes were within the normal range (supplemental Table 1,
available on the Blood Web site; see the Supplemental Materials
link at the top of the online article), indicating the absence of
significant anemia in the peripheral blood. We next characterized
hematopoietic properties of Fancd2?/?bone marrow cells. Both
Fancd2?/?and wild-type mice had equivalent numbers of total
nucleated bone marrow cells (data not shown). Compared with
wild-type controls, bone marrow cells of 3- to 8-month-old
1?Lineage?(KSL) cells (P ? .0001), a population enriched for
short- and long-term HSCs (Figure 1A-B). In contrast, Fancc?/?
mice had a KSL compartment comparable with their wild-type
littermates (P ? .05; Figure 1B). CD34 staining of KSL cells also
showed a significant reduction in long-term HSC-enriched
CD34?KSL cells in Fancd2?/?bone marrow (P ? .0001; Figure
1C). A thorough comparison between young and aged mice found
that even 3-week-old Fancd2?/?mice had a smaller KSLpool than
wild-type controls (supplemental Figure 1), suggesting that this
Figure 1. Fancd2?/?mice have a smaller KSL stem and progenitor pool. (A) FACS profiles after KSL staining of the bone marrow cells from Fancd2 mutant mice and
wild-type littermate controls. The percentage of the KSL gate is referring to the proportion of KSL cells in the whole nucleated bone marrow. To confirm the purity of
double-sorted KSLcells, 3000 cells were analyzed for each genotype. (B) Quantification of hematopoietic KSLstem and progenitor frequencies in the bone marrow of FAmice
(Fancd2?/?or Fancc?/?) and wild-type controls. All the mice used were between 3 and 8 months of age. In the Fancd2?/?group, n ? 19 for Fancd2?/?mice and n ? 12 for
Fancd2?/?littermate controls; in the Fancc?/?group, n ? 6 for Fancc?/?mice and n ? 4 for Fancc?/?littermate controls. NS denotes not significant. (C) Quantification of
CD34?KSL hematopoietic stem-cell frequencies in the bone marrow of Fancd2?/?mice or wild-type controls. N ? 6 for Fancd2?/?mice and n ? 7 for Fancd2?/?mice.
(D) Quantification of CLP frequencies in the bone marrow of Fancd2?/?mice or wild-type controls. N ? 5 for Fancd2?/?mice and n ? 3 for Fancd2?/?mice.
5142 ZHANG et al BLOOD, 9 DECEMBER 2010?VOLUME 116, NUMBER 24
defect might be generated in early development. KSL frequencies
in Fancd2?/?mice appeared to stabilize after 1 month of age and
did not decline further even in aged mice as old as 12 months.
To evaluate the effects of Fancd2 deficiency on defined
hematopoietic progenitor populations, we first examined the com-
mon myeloid progenitors and granulocyte-macrophage progenitors
of each population in Fancd2?/?mice was slightly, but nonsignifi-
cantly, lower than their counterpart in wild-type mice. However, we
did notice that 20% of Fancd2?/?mice displayed a marked
reduction in the frequency of CMPs or GMPs. Further analysis
found that CLPs are more than 50% fewer in Fancd2?/?mice than
those in the wild-type controls (P ? .01; Figure 1D). Thus, loss of
Fancd2 resulted in a smaller HSC pool and reduced lymphoid
progenitor frequencies. Taking into account that progenitor cell
numbers can be reduced in FA patients before the onset of overt
bone marrow failure,9,35the fact that Fancd2?/?mice show
hematopoietic defects, even in the absence of clinical anemia, is
congruent with the human condition.
Fancd2?/?bone marrow suffered functional loss of
hematopoietic stem and progenitor cells and had a
compromised marrow environment
To assess the functional properties of Fancd2?/?HSCs, we used a
long-term in vivo competitive repopulation assay and transplanted
bone marrow cells from either Fancd2?/?or Fancc?/?mice into
lethally irradiated FA mice (Fancd2?/?donor cells into Fancc?/?
recipients or vice versa), along with an equal number of cells from
ROSA26Tg/Omice, which are hemizygous for the ROSA26 trans-
genic strain and have an intact FA pathway. Like wild-type mice,
ROSA26Tg/Omice do not have any overt phenotypes and the
transgene is used simply as a genetic marker. Each different strain
(Fancd2?/?, Fancc?/?, or ROSA26Tg/O) has a unique genotype that
can be distinguished from one another by qPCR. Analysis of
peripheral blood 7-9 months posttransplantation revealed a dra-
matic reduction in donor-derived FA cells. Starting from an initial
input ratio of 1:1 (FA: ROSA26Tg/O), the ratio of donor-derived FA
cells vs. donor-derived ROSA26Tg/Ocells in the peripheral blood
dropped to 1:20 in the case of Fancd2?/?donor and 1:7 in the case
of Fancc?/?donor cells (Figure 2A). Together, these data show
compromised repopulating capacity by both Fancd2?/?and
Fancc?/?bone marrow cells.
Next, we measured the frequency of Fancd2?/?primitive
progenitors in vitro using the CAFC assay. In this assay, freshly
isolated bone marrow cells were used to establish an adherent
stromal/feeder layer, which comprised endothelial cells, fibro-
blasts, and adipocytes. The stromal layer provides a microenvi-
ronment that supports proliferation and differentiation of HSCs.
When plated on wild-type bone marrow–derived feeder/stromal
layers, Fancd2?/?bone marrow cells formed fewer than half the
number of colonies generated by Fancd2?/?controls, with an
average CAFC frequency of only 1/34 000 cells, compared with
1/16 000 cells in the wild type (P ? .005; Figure 2B-C). The
reduction of CAFC frequency in Fancd2?/?bone marrow cells
reflects the change in hematopoietic stem and progenitor numbers
in these animals, consistent with the observed reduction of KSL
cells. Along the same line, a CFU-S assay also confirmed that
Fancd2?/?bone marrow cells formed 65% fewer macroscopic
splenic colonies than wild-type controls (supplemental Figure 3),
suggesting a reduced number and function of progenitors in
To our surprise, we also found that Fancd2?/?bone marrow–
derived feeder layer cells were less supportive of progenitor growth
than wild-type controls in the CAFC assay. Both wild-type and
mutant marrow cells formed significantly fewer cobblestone colo-
nies when plated onto mutant feeders. In a direct comparison, the
CAFC frequency of wild-type marrow was only 1/27 000 on
Fancd2?/?feeders, compared with 1/16 000 on Fancd2?/?feeder
cells (P ? .05; Figure 2C). Similarly, Fancd2?/?bone marrow had
the lowest colony-forming frequency (1/74 000) when plated on
Fancd2?/?feeders. Collectively, these results suggest that Fancd2
deficiency affects not only the stem cells directly, but also causes a
defective marrow microenvironment.
Figure 2. Hematopoietic defects in Fancd2?/?mice. (A) In vivo competitive
repopulation of mixed FA (Fancc?/?or Fancd2?/?) and ROSA26Tg/Obone marrow
cells. Quantitative real-time PCR (qPCR) analyses were performed to evaluate
donor contribution to the peripheral blood cells from each donor (7 or 9 months
posttransplantation for Fancd2?/?or Fancc?/?donors, respectively). Three
independent qPCR analyses were performed for each sample, and results from
5 animals were pooled together for each experimental group. P values were
calculated by the 2-tailed, paired Student t test. Error bars represent SEM.
(B) Representative picture for CAFC assay. The arrow indicates cobblestone
colony. The image was acquired on an Axiovert 200 inverted microscope with an
AxioCam MRc color camera at room temperature using AxioVision Release 4.8
software (Carl Zeiss MicroImaging). Original magnification ?100 (with 10?
objective lens). (C) Quantification of CAFC results. P values were calculated by
2-tailed, paired Student t test.
Fancd2 MAINTAINS QUIESCENCEAND MARROW ENVIRONMENT5143BLOOD, 9 DECEMBER 2010?VOLUME 116, NUMBER 24
Fancd2?/?KSL cells showed an abnormal cell-cycle status
We next examined the cell-cycle status of Fancd2?/?KSL cells
using DNA-binding dye Hoechst 33342. The proportion of
Fancd2?/?KSL cells in the S-G2-M phases of the cell cycle was
markedly higher than that in wild-type KSL cells (Figure 3A-B).
Nearly twice as many cells had G2DNA content, indicating a
spontaneous delay in S/G2/M. Importantly, this late S-phase/G2-M
delay is a hallmark of FA cells in culture and has been used
diagnostically.36Similar trends were also seen in Fancc?/?KSL
cells, although the difference between Fancc?/?KSL cells and
To confirm that Fancd2?/?stem cells were less quiescent, we
combined the above cell-cycle analysis approach with antibody
staining for Ki-67, which is not expressed by quiescent cells.
Because cells in both G0and G1phases of the cell cycle have 2c
DNAcontent by Hoechst 33342 staining, the use of Ki-67 antibody
allowed us to distinguish G0(negative Ki-67 staining) from G1cells
(positive Ki-67 staining). We found that the average G0proportion
of Fancd2?/?KSLcells was 25%, significantly lower than the 45%
observed in the wild-type counterparts (P ? .0001; Figure 3B). A
cells showed a 2-fold higher frequency of S/G2/M cells (P ? .05)
and a significantly lower frequency of G0cells than Fancd2?/?
CD34?KSL cells (P ? .01; Figure 3C). In some extreme cases,
such as that shown in Figure 3D, as few as 12% of Fancd2?/?KSL
cells were in G0, compared with 47% G0frequency in KSL cells
from a littermate wild-type control. These results demonstrate that
Fancd2 deficiency led to increased cell-cycle entry and the loss of
quiescence in HSCs.
Resveratrol treatment partially corrected the hematopoietic
defects in Fancd2?/?mice
The abnormal quantitative phenotypes described in this Fancd2?/?
murine model provide an opportunity to directly ascertain the
therapeutic effects of drugs on HSCs.
that this compound could enhance hematopoiesis in Fancd2?/?
mice. Fancd2?/?mice and their wild-type littermates were treated
with either control diet or diet supplemented with resveratrol.
Treatment began at 1 month of age and continued for up to
9 months. Experimental mice showed no signs of weight loss or
drug toxicity. Flow cytometric analysis showed no difference in KSL
frequencies between resveratrol-treated and placebo-treated mice
(Figure 4A). However, cell cycle analysis revealed a 27% increase
in the G0proportion of KSL cells in resveratrol-treated Fancd2?/?
mice (Figure 4B-D). Importantly, CFU-S assays with whole bone
marrow demonstrated that the frequency of primitive spleen
colony-forming cells in resveratrol-treated Fancd2?/?mice was
significantly improved, compared with placebo-treated controls
(P ? .02; Figure 4E). The numbers of CFU-S doubled after
resveratrol treatment, and many were in the range obtained from
normal bone marrow. These data suggest that resveratrol can
partially correct the hematopoietic defects of Fancd2?/?mice.
We next tested whether resveratrol could affect the function of
the bone marrow stromal cells in CAFC assays. Fancd2?/?bone
marrow–derived feeder/stromal layers were established in the
absence or presence of resveratrol. When wild-type bone marrow
cells were plated on top of the different feeders, CAFC frequencies
on resveratrol-treated Fancd2?/?feeder layers were 42% higher
Figure 3. Fancd2?/?KSL cells have abnormal cell
cycle status. (A) Representative cell-cycle profiles of
KSL cells from a Fancd2?/?mouse and its wild-type
littermate control. DNA content was measured with
Hoechst 33342. Data were analyzed with FlowJo (TreeS-
tar) using the Dean-Jett-Fox model for the quantification
of each cell-cycle phase. (B) Pooled results from cell
cycle analysis on KSL cells. N ? 9 for each group.
cells. N ? 3 for each group. (D) Representative picture
after costaining for DNA content (Hoechst 33342) and
Ki67 expression in KSL cells from a Fancd2?/?mouse
and its wild-type littermate control. Percentages for each
gate were denoted.
5144ZHANG et alBLOOD, 9 DECEMBER 2010?VOLUME 116, NUMBER 24
than those on placebo-treated Fancd2?/?feeder layers (P ? .05,
2-tailed paired Student t test; n ? 3), indicating that resveratrol
treatment alleviates the defects of Fancd2?/?stromal cells in
supporting hematopoiesis. There was no difference in CAFC
frequencies between resveratrol-treated wild-type bone marrow–
derived stromal layers and placebo-treated wild-type controls (data
not shown), suggesting that the beneficial effect of resveratrol on
Fancd2?/?feeder function is not due to a general improvement of
colony formation on all the feeder layers.
Given that FA cells are hypersensitive to reactive oxygen
species (ROS),19,37we therefore tested the hypothesis that ROS
levels are increased in KSL cells from Fancd2?/?mice. Double-
Figure 4. Resveratrol partially corrects hematopoietic defects in Fancd2?/?mice. (A) KSLfrequency was not significantly changed in either Fancd2?/?or Fancd2?/?mice
after resveratrol (RV) treatment. (B) Statistical quantification of the cell-cycle analysis on KSL cells in resveratrol- versus placebo-treated mice. N ? 3 for each group.
(C) Representative cell cycle profiles of a placebo-treated Fancd2?/?mouse, its resveratrol-treated Fancd2?/?littermate control, and placebo-treated wild-type control. Cells
are stained for KSL and DNA content (Hoechst 33342). Data were analyzed with FlowJo using Dean-Jett-Fox model for the quantification of each cell cycle phase.
(D) Representative picture after a costaining for DNA content (Hoechst 33342) and Ki67 expression in KSL subsets of the bone marrow cells from placebo- or
resveratrol-treated Fancd2?/?mice and a placebo-treated wild-type littermate control. Percentages for each gate were denoted. (E) Resveratrol treatment significantly
improved the CFU-S–forming capacity of Fancd2?/?bone marrow cells. Data represent 3 donors for each group with 3 (or 2 in 1 case) recipients for each donor.
Fancd2 MAINTAINS QUIESCENCEAND MARROW ENVIRONMENT5145 BLOOD, 9 DECEMBER 2010?VOLUME 116, NUMBER 24
sorted KSLcells were stained with carboxy-H2DCFDAto measure
the intracellular concentrations of ROS. Under our experimental
conditions, we failed to detect any difference in ROS levels
between Fancd2?/?KSL cells and wild-type controls (Figure 5A).
Because resveratrol may affect production of and/or hypersensi-
tivity to proapoptotic cytokine TNF-? in the hematopoietic micro-
environment,14,38,39we measured the levels of apoptosis in
Fancd2?/?and wild-type bone marrow cells. Annexin V and
7-aminoactinomycin D (7-AAD) staining revealed no significant
gross increase in early apoptotic cells, defined as annexin V
positive and 7-AAD negative, in Fancd2?/?whole bone marrow
cells, compared with wild-type controls. Further analysis found
that Fancd2?/?KSL cells have a higher, but nonsignificant,
proportion of cells undergoing apoptosis than wild-type controls
(Figure 5B). It is possible that the increase of apoptosis level is
insufficient to be detectable under these experimental condi-
tions, or that the lack of statistical significance was a function of
To directly test whether resveratrol could influence cytokine
response in FAcells, we used a recently developed system in which
the overproduction of TNF-? by human FANCC-deficient and
tively measured by ELISA.34This system was suitable for small-
molecule screening and permitted us to test the capacity of
resveratrol to influence that phenotype in FANCC-deficient cells. In
the absence of resveratrol, imidazoquinoline resiquimod R848
induced the overexpression of TNF-? in FANCC-deficient THP1
cells, as quantified by TNF-? ELISA. Treatment of these cells with
10?M or 100?M resveratrol suppressed TNF-? production in
these cells (Figure 5C). These results demonstrate that resveratrol
can influence a quantitative hematopoietic defect in cells harboring
a disrupted FA pathway. Although this effect appeared not to be
specific for FA cells, as shown by Figure 5C, the benefit from
resveratrol treatment could be more significant for FA cells, since
these cells are more sensitive to TNF-?.
In this work, we characterized hematopoietic phenotypes in
Fancd2?/?mice and found multiple defects, including stem-cell
and progenitor loss in early development, compromised functional
capacity of HSCs, and a less supportive marrow environment. This
work highlights the critical role of Fancd2 in stem-cell survival and
It should be noted that others have reported Fancc-mutant mice
to display progenitor defects when on a C57/B6 background.18
Nonetheless, in a direct comparison of Fancd2?/?and Fancc?/?
mice on the same 129S4 mouse strain, we show here that
Fancd2?/?mice have markedly lower numbers of KSL stem and
progenitor cells, a phenotype that might be formed in early
development and not shared by Fancc?/?mice. Consistent with our
findings, developmental abnormalities have also been observed in
Fancd2-knockdown zebrafish.40These data support the notion that
Fancd2 might have an especially critical function in some early
developmental processes, including hematopoiesis.2In humans,
FANCD2 could be essential for embryonic survival, considering
that all the FANCD2 mutations identified in human patients are
hypomorphic.24The fact that Fancc?/?mice have a normal-sized
KSL stem and progenitor pool could imply that nonubiquitinated
Fancd2 has indispensable roles in these developmental processes,
as some have recently speculated.2
An important finding of this study is that Fancd2?/?mice have
a compromised marrow environment that is less supportive for
stem cell population. Similarly, it has recently been reported that
Fancg?/?mesenchymal stem/progenitor cells have defects in their
etic stem/progenitor cells.41Considering the crucial function of
marrow environment in balancing stem cell self-renewal and
differentiation, we propose this phenotype is very likely to
contribute to the pathogenesis of FA.
Figure 5. ROS levels, apoptosis, and cytokine re-
sponse in Fancd2?/?and wild-type bone marrow
cells. (A) Fluorescence intensity in KSL subsets of
Fancd2?/?and wild-type bone marrow after carboxy-
H2DCFDA staining for intracellular ROS. Oxidation of
carboxy-H2DCFDA by ROS generated fluorescence de-
tectable by flow cytometry. (B) Quantification of early
apoptotic KSL cells after 7-AAD, annexin V, and KSL
staining. Data represent 7 mice for each group. NS
induced TNF-? overproduction in Fancc-shRNA–treated
in triplicates on a 96-well plate.
5146 ZHANG et alBLOOD, 9 DECEMBER 2010?VOLUME 116, NUMBER 24
Despite the marked reduction in HSC and progenitor numbers
and the reduced capacity for stem-cell function, Fancd2?/?mice
did not show spontaneous anemia in their peripheral blood and
maintained relatively normal blood counts. How do Fancd2?/?
mice maintain the homeostasis in their blood system? We have
observed in this work that HSCs and progenitors in Fancd2?/?
mice failed to maintain their quiescence and, hence, enter the cell
cycle more frequently. We speculate that this could compensate for
consequences from the loss in HSC number and function. How-
ever, in doing so, the animals could face a long-term risk of
stem-cell exhaustion that could eventually lead to bone marrow
failure, given the compelling evidence that enhanced cell cycle
entry causes stem cell exhaustion and leads to long-term loss of
stem cell function.42-44
The loss of quiescence and reduction in the KSL pool along
with a dysfunctional marrow stroma converge to create several
quantitative phenotypes ideally suited for pharmacological studies.
Not surprisingly, the developmental defect of reduced KSL num-
bers could not be corrected by postnatal treatment with resveratrol.
Encouragingly, however, this Sirt1-mimetic compound showed
measurable benefit in functional hematopoietic assays both in vivo
(CFU-S, cell cycle profile) and in vitro (CAFC). By improving the
function of HSCs and progenitors, resveratrol could relieve the
pressure of enhanced cell cycle entry faced by those cells and has
the potential to slow down the path toward stem cell exhaustion.
There are several possible mechanisms by which resveratrol
could improve hematopoietic function in Fancd2?/?mice. Because
SirT1 is expressed at high levels in HSCs,45resveratrol could
stimulate SirT1 activity and directly enhance SirT1-mediated DNA
repair.29,30However, the function of SirT1 in HSC remains elusive.
Further work needs to be done to determine, among the myriad of
SirT1 target proteins, which protein is specifically influenced by
resveratrol treatment to enhance hematopoiesis. Alternatively,
resveratrol might alter HSC activity through SirT1-independent
mechanisms.46We did find that resveratrol treatment could sup-
press TNF-? production in FA-deficient cells. It remains to be
determined whether resveratrol could have a similar effect on
TNF-? production in FA cells in vivo. It is also important to
emphasize that our data do not address whether the effects of
resveratrol are specific to only FA or whether hematopoietic stem
cells from wild-type mice also respond to this compound. Given
the fact that resveratrol has shown beneficial effects in many
non-FAphenotypes, it appears unlikely that it acts in an FA-specific
way.47,48Nonetheless, it partially normalized important hematopoi-
etic parameters in our Fancd2?/?mouse model and hence should
be further pursued therapeutically.
plete, more powerful Sirt1-stimulating compounds have been
recently developed and may prove more potent in achieving
hematopoietic enhancement.49In the meantime, resveratrol itself
may be suitable for clinical trials in FA.
We thank Pamela Canady, Mandy Boyd, and Dorian LaTocha at the
Oregon Health & Science University flow cytometry core for
fluorescence activated cell sorting.
This work was supported by grants 1P01HL48546, HL077818,
HL069133, and 1R01CA138237 and by the VeteransAffairs Merit
Contributions: Q.-S.Z. designed the study, performed research,
analyzed and interpreted data, and wrote the manuscript; L.M.-L.,
L.E., and K.W.-S. performed research; A.W.D. and D.C.G. de-
signed the study and wrote the manuscript; P.A. and R.K.R.
performed the research; W.H.F. interpreted data and wrote the
manuscript; G.C.B. designed TNF-? assays, interpreted data, and
wrote the manuscript; and M.G. designed the study, analyzed and
interpreted data, and wrote the manuscript.
Conflict-of-interest disclosure: The authors declare no compet-
ing financial interests.
Correspondence: Qing-Shuo Zhang, Oregon Stem Cell Center,
Oregon Health & Science University, 3181 SW Sam Jackson Park
Rd, Portland, OR 97239; e-mail: email@example.com.
1. Moldovan GL, D’AndreaAD. How the Fanconi
anemia pathway guards the genome. Annu Rev
2. Wang W. Emergence of a DNA-damage re-
sponse network consisting of Fanconi anaemia
and BRCAproteins. Nat Rev Genet. 2007;8(10):
3. Kutler DI, Singh B, Satagopan J, et al.A20-year
perspective on the International FanconiAnemia
Registry (IFAR). Blood. 2003;101(4):1249-1256.
4. Prasher JM, LalaiAS, Heijmans-Antonissen C, et
al. Reduced hematopoietic reserves in DNAinter-
strand cross-link repair-deficient Ercc1?/? mice.
EMBO J. 2005;24(4):861-871.
5. Rossi DJ, Bryder D, Seita J, NussenzweigA,
Hoeijmakers J, Weissman IL. Deficiencies in
DNAdamage repair limit the function of haemato-
poietic stem cells with age. Nature. 2007;
6. Ruzankina Y, Pinzon-Guzman C,AsareA, et al.
Deletion of the developmentally essential gene
ATR in adult mice leads to age-related pheno-
types and stem cell loss. Cell Stem Cell. 2007;
7. Bagby GC,Alter BP. Fanconi anemia. Semin He-
8. Du W,Adam Z, Rani R, Zhang X, Pang Q. Oxida-
tive stress in Fanconi anemia hematopoiesis and
disease progression. Antioxid Redox Signal.
9. Muller LU, Williams DA. Finding the needle in the
hay stack: Hematopoietic stem cells in Fanconi
anemia. Mutat Res. 668(1-2):141-149, 2009.
10. Dufour C, CorcioneA, Svahn J, et al. TNF-alpha
and IFN-gamma are overexpressed in the bone
marrow of Fanconi anemia patients and TNF-
alpha suppresses erythropoiesis in vitro. Blood.
11. Rosselli F, Sanceau J, Gluckman E, Wietzerbin J,
Moustacchi E.Abnormal lymphokine production:
a novel feature of the genetic disease Fanconi
anemia. II. In vitro and in vivo spontaneous over-
production of tumor necrosis factor alpha. Blood.
12. Schultz JC, Shahidi NT. Tumor necrosis factor-
alpha overproduction in Fanconi’s anemia. Am J
13. Rathbun RK, Faulkner GR, Ostroski MH, et al.
Inactivation of the Fanconi anemia group C gene
augments interferon-gamma–induced apoptotic
responses in hematopoietic cells. Blood. 1997;
14. Li J, Sejas DP, Zhang X, et al. TNF-alpha induces
leukemic clonal evolution ex vivo in Fanconi ane-
mia group C murine stem cells. J Clin Invest.
15. Li X, Yang Y, Yuan J, et al. Continuous in vivo in-
fusion of interferon-gamma (IFN-gamma) prefer-
entially reduces myeloid progenitor numbers and
enhances engraftment of syngeneic wild-type
cells in Fancc?/? mice. Blood. 2004;104(4):
16. Taniguchi T, D’AndreaAD. Molecular pathogene-
sis of Fanconi anemia: recent progress. Blood.
17. Battaile KP, Bateman RL, Mortimer D, et al. In
vivo selection of wild-type hematopoietic stem
cells in a murine model of Fanconi anemia.
18. Carreau M, Gan OI, Liu L, Doedens M, Dick JE,
Buchwald M. Hematopoietic compartment of Fan-
coni anemia group C null mice contains fewer
lineage-negative CD34? primitive hematopoietic
cells and shows reduced reconstruction ability.
Exp Hematol. 1999;27(11):1667-1674.
19. Haneline LS, Gobbett TA, Ramani R, et al. Loss
of FancC function results in decreased hemato-
poietic stem cell repopulating ability. Blood. 1999;
20. Zhang QS, Eaton L, Snyder ER, et al. Tempol
protects against oxidative damage and delays
Fancd2 MAINTAINS QUIESCENCEAND MARROW ENVIRONMENT5147 BLOOD, 9 DECEMBER 2010?VOLUME 116, NUMBER 24
epithelial tumor onset in Fanconi anemia mice.
Cancer Res. 2008;68(5):1601-1608.
21. Houghtaling S, Timmers C, Noll M, et al. Epithe-
lial cancer in Fanconi anemia complementation
group D2 (Fancd2) knockout mice. Genes Dev.
22. Blom E, van de Vrugt HJ, de Winter JP,Arwert F,
Joenje H. Evolutionary clues to the molecular
function of fanconi anemia genes. Acta Haema-
23. Timmers C, Taniguchi T, Hejna J, et al. Positional
cloning of a novel Fanconi anemia gene,
FANCD2. Mol Cell. 2001;7(2):241-248.
24. Kalb R, Neveling K, Hoehn H, et al. Hypomorphic
mutations in the gene encoding a key Fanconi
anemia protein, FANCD2, sustain a significant
group of FA-D2 patients with severe phenotype.
Am J Hum Genet. 2007;80(5):895-910.
25. Finkel T, Deng CX, Mostoslavsky R. Recent
progress in the biology and physiology of sirtuins.
26. Guarente L, Picard F. Calorie restriction—the
SIR2 connection. Cell. 2005;120(4):473-482.
27. Firestein R, Blander G, Michan S, et al. The
SIRT1 deacetylase suppresses intestinal tumori-
genesis and colon cancer growth. PLoS ONE.
28. Howitz KT, Bitterman KJ, Cohen HY, et al. Small
molecule activators of sirtuins extend Saccharo-
29. Oberdoerffer P, Michan S, McVay M, et al. SIRT1
redistribution on chromatin promotes genomic
stability but alters gene expression during aging.
30. Wang RH, Sengupta K, Li C, et al. Impaired DNA
damage response, genome instability, and tu-
morigenesis in SIRT1 mutant mice. Cancer Cell.
31. Gao X, Xu YX, Divine G, Janakiraman N,
Chapman RA, Gautam SC. Disparate in vitro and
in vivo antileukemic effects of resveratrol, a natu-
ral polyphenolic compound found in grapes.
J Nutr. 2002;132(7):2076-2081.
32. Willenbring H, BaileyAS, Foster M, et al. My-
elomonocytic cells are sufficient for therapeutic
cell fusion in liver. Nat Med. 2004;10(7):744-748.
33. WilsonA, Murphy MJ, Oskarsson T, et al. c-Myc
controls the balance between hematopoietic stem
cell self-renewal and differentiation. Genes Dev.
34. Vanderwerf SM, Svahn J, Olson S, et al. TLR8-
dependent TNF-(alpha) overexpression in Fan-
coni anemia group C cells. Blood. 2009;114(26):
35. AuerbachAD, Liu Q, Ghosh R, Pollack MS,
Douglas GW, Broxmeyer HE. Prenatal identifica-
tion of potential donors for umbilical cord blood
transplantation for Fanconi anemia. Transfusion.
36. BechtoldA, Friedl R, Kalb R, et al. Prenatal exclu-
sion/confirmation of Fanconi anemia via flow cy-
tometry: a pilot study. Fetal Diagn Ther. 2006;
37. Joenje H,Arwert F, ErikssonAW, de Koning H,
OostraAB. Oxygen-dependence of chromosomal
aberrations in Fanconi’s anaemia. Nature. 1981;
38. Sejas DP, Rani R, Qiu Y, et al. Inflammatory reac-
tive oxygen species-mediated hemopoietic sup-
pression in Fancc-deficient mice. J Immunol.
39. Zhang X, Sejas DP, Qiu Y, Williams DA, Pang Q.
Inflammatory ROS promote and cooperate with
the Fanconi anemia mutation for hematopoietic
senescence. J Cell Sci. 2007;120(Pt 9):1572-
40. Liu TX, Howlett NG, Deng M, et al. Knockdown of
zebrafish Fancd2 causes developmental abnor-
malities via p53-dependent apoptosis. Dev Cell.
41. Li Y, Chen S, Yuan J, et al. Mesenchymal stem/
progenitor cells promote the reconstitution of ex-
ogenous hematopoietic stem cells in Fancg?/?
mice in vivo. Blood. 2009;113(10):2342-2351.
42. Orford KW, Scadden DT. Deconstructing stem
cell self-renewal: genetic insights into cell-cycle
regulation. Nat Rev Genet. 2008;9(2):115-128.
43. Hock H, Hamblen MJ, Rooke HM, et al. Gfi-1 re-
stricts proliferation and preserves functional in-
tegrity of haematopoietic stem cells. Nature.
44. Zhang J, Grindley JC, Yin T, et al. PTEN main-
tains haematopoietic stem cells and acts in lin-
eage choice and leukaemia prevention. Nature.
45. Chambers SM, Boles NC, Lin KY, et al. Hemato-
poietic fingerprints: an expression database of
stem cells and their progeny. Cell Stem Cell.
46. Zhang J. Resveratrol inhibits insulin responses in
a SirT1-independent pathway. Biochem J. 2006;
47. Sakata Y, Zhuang H, Kwansa H, Koehler RC,
Dore S. Resveratrol protects against experimen-
tal stroke: putative neuroprotective role of heme
oxygenase 1. Exp Neurol. 2010;224(1):325-329.
48. Yao J, Wang JY, Liu L, et al.Antioxidant effects of
resveratrol on mice with DSS-induced ulcerative
colitis. Arch Med Res. 2010;41(4):288-294.
49. Milne JC, Lambert PD, Schenk S, et al. Small
molecule activators of SIRT1 as therapeutics for
the treatment of type 2 diabetes. Nature. 2007;
5148 ZHANG et al BLOOD, 9 DECEMBER 2010?VOLUME 116, NUMBER 24