HEMATOPOIESISAND STEM CELLS
*Olga Bromberg,1,2*Benjamin J. Frisch,1,3Jonathan M. Weber,1,2Rebecca L. Porter,1,2Roberto Civitelli,4and
Laura M. Calvi1,2
1Endocrine Division, Department of Medicine,2Center for Musculoskeletal Research, and3Department of Pathology and Laboratory Medicine, University of
Rochester School of Medicine and Dentistry, Rochester, NY; and4Division of Bone and Mineral Diseases, Washington University, St Louis, MO
Hematopoietic stem cell (HSC) regulation
is highly dependent on interactions with
the marrow microenvironment. Contro-
versy exists on N-cadherin’s role in sup-
port of HSCs. Specifically, it is unknown
whether microenvironmental N-cadherin
tecture and for hematopoiesis. To deter-
mine whether osteoblastic N-cadherin is
required for HSC regulation, we used a
genetic murine model in which deletion of
Cdh2, the gene encoding N-cadherin, has
been targeted to cells of the osteoblastic
lineage. Targeted deletion of N-cadherin
type, ultimately characterized by de-
creased mineralized bone, but no differ-
ence in steady-state HSC numbers or
function at any time tested, and normal
recovery from myeloablative injury. Inter-
mittent parathyroid hormone (PTH) treat-
ment is well established as anabolic to
bone and to increase marrow HSCs
Lack of osteoblastic N-cadherin did not
block the bone anabolic or the HSC ef-
strates that osteoblastic N-cadherin is
not required for regulation of steady-state
hematopoiesis, HSC response to myelo-
ablation, or for rapid expansion of HSCs
through intermittent treatment with PTH.
The molecular signals that mediate regulatory microenvironmental
osteoblastic-hematopoietic interactions provide potential therapeu-
tic targets for hematopoietic stem cell (HSC) manipulation1but are
still largely unknown. Based on the essential role of cadherins in
fate specification of germline stem cells2and the close proximity of
N-cadherin–expressing osteoblastic cells to HSCs,3-6it was ini-
tially proposed that N-cadherin may provide HSCs with instructive
interactions with their niche. However, much debate was subse-
quently raised by reports questioning N-cadherin expression by
HSCs,7whereas others suggested that different N-cadherin levels
characterize functionally distinct HSC populations,5,8,9which may
respond to niche manipulation.10Moreover, inducible global ge-
netic N-cadherin deletion lacks hematopoietic defects,11but knock-
down of N-cadherin in HSCs suppresses their long-term engraft-
ment.12Whereas in HSCs N-cadherin, when present, is expressed
at very low levels,13osteoblastic cells express N-cadherin at several
differentiation stages, both in immature and mature cells.3,14
(Cdh2) is embryonically lethal,15hemizygous mice already display
osteoblastic defects, having accentuated bone loss with ovariec-
tomy.16Moreover, recent data have suggested differential roles of
N-cadherin at various stages of osteoblastic differentiation, with
actions of N-cadherin both on osteogenic commitment as well as at
terminal differentiation.14In this work, our goal was to determine
whether targeted deletion of Cdh2 in maturing osteoblasts alters the
BM microarchitecture, HSC number, and function in homeostasis
and disrupts the action of parathyroid hormone (PTH) on the
Col2.3-Cre mice expressing the Cre-recombinase under the control of the
2.3-kb fragment of the ?1(I) collagen gene promoter were kindly provided
by Dr Gerard Karsenty.17Mating of Col2.3-Cre with Cdh2flmice (generated
in Dr Glenn L. Radice’s laboratory, Thomas Jefferson University, Philadel-
phia, PA)18resulted in mice carrying the Col2.3-Cre?Cadh2fl/flgenotype,
which had specific inactivation of N-cadherin in cells of the osteoblastic
lineage, designated as OB-NCadh for the rest of the manuscript.14This line
was maintained in the C57bl/6 background, and expression of the CD45.2
congenic marker was confirmed. Wild-type (WT) mice expressing the
CD45.1 congenic marker (B6.SJL-PtprcaPep3b/BoyJ CD45.1) were pur-
chased from The Jackson Laboratory. Genotyping was performed as
previously described.14B6.129(Cg)-Tg(CAG-Bgeo/GFP)21Lbe/J (Z/EG)
mice were also purchased from The Jackson Laboratory and were
genotyped according to The Jackson Laboratory’s recommendations. Mice
were maintained under microisolator technique. All experiments on mice
were approved by the Institutional Animal Care and Use Committee at the
University of Rochester School of Medicine.
Osteoblastic cell collection
Osteoblastic cells were isolated using collagenase digestion and magnetic
separation based on CD45 expression as previously described.19Osteoblas-
tic cells from adult mice were obtained from the long bones of the
hindlimbs and forelimbs, as well as from calvaria. Osteoblastic cells from
neonatal mice were obtained from calvaria.
Analysis of mRNA abundance by real-time RT-PCR
Total mRNA was extracted using the RNeasy kit (QIAGEN) according to
the manufacturer’s instructions. Total mRNA was then reverse-transcribed
Submitted September 2, 2011; accepted May 7, 2012. Prepublished online as
Blood First Edition paper, May 17, 2012; DOI 10.1182/blood-2011-09-377853.
*O.B. and B.J.F. contributed equally to this study.
There is an Inside Blood commentary on this article in this issue.
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.
© 2012 by TheAmerican Society of Hematology
303 BLOOD, 12 JULY 2012?VOLUME 120, NUMBER 2
For personal use only.on November 5, 2015. by guest
to produce cDNA using the Quantitect Reverse Transcription kit (QIA-
GEN). cDNA was amplified using MyiQ Single Color PCR detection
systems and MyIQ Version 1.0.410 software under the following condi-
tions: 95°C for 3 minutes followed by 40 cycles of 95°C for 15 seconds and
58°C for 30 seconds. Data were analyzed using the relative standard curve
method, normalized to ?-actin: ?-actin, 5? primer GCCACTGCCGCATC-
CTCTT and 3? primer GGAACCGCTCGTTGCCAATAG; and Cdh2, 5?
primer ATTCAGCACCCACCTCAGTC and 3? primer TCCGCCT-
Histology and immunohistochemistry
Harvested hindlimbs were fixed, decalcified, and processed as described.20
Histologic sections were stained with H&E to visualize morphology.
Paraffin-embedded sections were cut at 4-?m thickness. All slides were
deparaffinized and rehydrated to PBS (pH 7.4) and blocked in 5% normal
goat serum for 60 minutes. Blocking serum was removed and 1:50 dilution
of anti–N-cadherin antibody (IBLno. 18571) was applied overnight at 4°C.
The biotinylated secondary antibody goat anti–rabbit at 1:200 dilution
(Vector BA-1000) was then applied for 30 minutes at room temperature.
The alkaline phosphatase-based ABC-AP detection system (Vector AK-
5000) was applied for 30 minutes followed by Vector Red chromogen
(Vector SK-4100) for 20 to 30 minutes. Slides were counterstained with fast
green solution followed by 1% glacial acetic acid, dehydrated, and
coverslipped with cytoseal.
Histology slides were viewed at room temperature with a Bx41 upright
microscope (Olympus). Objectives used were UPlan Fl 4?/0.13, UPlan Fl
20?/0.50, and UPlan FLN 60?/0.90 (Olympus).All images were obtained
with a SPOT Insight 4 digital microscope camera and SPOT Version 6.7
The limbs were scanned on aViva CT40 (Scanco Medical) using a 55-kVp,
145-?Acurrent and a 300-ms integration time with a resolution of 12.5 ?m.
Trabecular analysis was conducted on a 1.25-mm region 50 ?m above the
growth plate in the femur and a 625-?m region 50 ?m below the growth
plate in the tibia. Cortical analysis was conducted 4.375 mm above/below
the growth plate of the femur/tibia for a distance of 375 ?m.
Flow cytometric analysis
BM mononuclear cells were obtained as previously described.21A total of
106to 107cells were then stained to identify Lineage?Sca1?c-kit?(LSK)
cells as previously described.21HSPCs were identified by the phenotypic
markers. Stained samples were analyzed on a LSR-II (BD Biosciences), and
results were quantified using Flowjo software (TreeStar). All antibodies
the Blood Web site; see the Supplemental Materials link at the top of the online
Blood and spleen analysis
Blood was collected by mandibular sampling, and samples were run
through the CBC-DIFF Veterinary Hematology System (HESKA) to obtain
platelet, white blood cells (WBCs), and hematocrit counts. Spleens were
collected in their entirety, weighed, and then mechanically disrupted to
obtain a total cell count.
Competitive transplantation assay
For experiments in OB-NCadh mice and littermate controls, BM cells
harvested as above for flow cytometric analysis from OB-NCadh and
control (NCadhfl/fl) mice (n ? 4-7 donor mice) was mixed with competi-
tor CD45.1 marrow cells at a ratio of 1:4 (for 6-month-old mice) or 1:2
(for 1-year-old and for 2- to 3-month-old mice treated with intermittent
PTH; donor/competitor) and 750 000 total cells were transplanted into
each CD45.1 recipient mice (10 recipient mice per genotype). Recipient
mice received a split dose of radiation of 5 Gy each separated by
24 hours. The second dose of radiation occurred 1 to 2 hours before the
transplantation. For PTH-treated mice and controls, whole BM from
PTH and vehicle (VEH)–treated CD45.1 C57/BL6 mice (n ? 3 donor
mice per treatment group) was harvested as previously described21and
1:2 (donor/competitor), and 750 000 total cells were transplanted into
each CD45.2 recipient mice. The peripheral blood of transplanted mice
was sampled by mandibular bleeds at times indicated to monitor
engraftment. Blood was separated in a 2% solution of 5 ? 105molecular
weight dextran to precipitate the RBCs. The resulting supernatant
containing WBCs was analyzed by flow cytometric analysis to assess
expression of congenic hematopoietic markers. For secondary transplan-
tation, BM was harvested from competitively transplanted animals,
which contained a combination of CD45.1 and CD45.2 marrow
20 weeks after primary competitive repopulation. BM from 3 animals of
the same treatment group was pooled, and 750 000 cells were trans-
planted into irradiated CD45.2 recipient mice.
fromCD45.2 ata ratio
rPTH (1-34) was purchased from Bachem and resuspended in water to
400 ?g/mL. This solution was diluted 1:100 in sterile PBS and adminis-
tered intraperitoneally to WT 8- to 10-week-old C57/BL6 male mice or
control and experimental mice of the ages and sex designated at 40 ?g/kg
3 times daily for 10 days. Fifteen hours after the last injection, the mice
were killed. The left hind limb was harvested for micro-CT analysis and
histology. BM was collected from the right hind limb and used for
For quantitative assays, treatment groups were reported as mean plus or
minus SEM. Statistical analysis was performed using the 2-tailed Student
t test or 2-way ANOVA with Bonferroni Multiple Comparison posttest,
when multiple comparisons to control group were made. For contingency
analysis of engraftment, a threshold was set at less than or equal to 1%
engraftment, 2-sample test for equality of proportions was applied, and the
Fisher exact t test was used to calculate significance. Statistical significance
was denoted by P ? .05 (Prism Version 4.01 for Windows; GraphPad
Genetic deletion of osteoblastic N-cadherin increases
trabecular volume in adult male mice
Targeted deletion of Cdh2 in osteoblastic cells did not result in
any overt skeletal abnormalities. Specific expression of Cre
recombinase under the control of the 2.3Col promoter was
demonstrated by confocal microscopic imaging of frozen sec-
tions from 2.3Col-Cre?Z/EG?mice (Figure 1A) consistent with
previously published data.17We determined the levels of Cdh2
gene expression in a population of cells that was previously
determined to be enriched for osteoblastic cells.19We observed a
trend toward a decrease in the expression of Cdh2 in cells
isolated from OB-NCadh mice compared with those isolated
from WT littermate controls (Figure 1B-C). Cdh2 expression was
highly variable in WT controls probably because of a lack of purity in
the population of cells analyzed. This variability, coupled with the low
level of Cdh2 gene expression under normal conditions, results in our
loss of N-cadherin?cells lining the endosteal surface of bone in adult
did not exhibit an aberrant bone phenotype compared with WT
304 BROMBERG et al BLOOD, 12 JULY 2012?VOLUME 120, NUMBER 2
For personal use only.on November 5, 2015. by guest
B.J.F. and L.M.C. wrote the manuscript; and B.J.F., L.M.C., O.B.,
J.M.W., and R.C. discussed data and edited the manuscript.
Conflict-of-interest disclosure: The authors declare no compet-
ing financial interests.
Correspondence: Laura M. Calvi, Endocrine Division, Depart-
ment of Medicine, University of Rochester School of Medicine,
601 Elmwood Ave, Box 693, Rochester, NY 14642; e-mail:
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OSTEOBLASTIC Cdh2 IS DISPENSABLE FOR HSC SUPPORT 313BLOOD, 12 JULY 2012?VOLUME 120, NUMBER 2
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online May 17, 2012
2012 120: 303-313
Laura M. Calvi
Olga Bromberg, Benjamin J. Frisch, Jonathan M. Weber, Rebecca L. Porter, Roberto Civitelli and
and regulation of hematopoietic stem and progenitor cells
Osteoblastic N-cadherin is not required for microenvironmental support
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