Articles

https://doi.org/10.1038/s41591-018-0030-x

Corrected: Author Correction

Adrenergic nerve degeneration in bone marrow

drives aging of the hematopoietic stem cell niche
Maria Maryanovich 1,2, Ali H. Zahalka 1,2,7, Halley Pierce1,2,7, Sandra Pinho 1,2,3, Fumio Nakahara ,
1,2

Noboru Asada1,2,6, Qiaozhi Wei1,2, Xizhe Wang1,2, Paul Ciero1,2, Jianing Xu4, Avigdor Leftin5 and
Paul S. Frenette 1,2,3*

Aging of hematopoietic stem cells (HSCs) is associated with a decline in their regenerative capacity and multilineage differen-
tiation potential, contributing to the development of blood disorders. The bone marrow microenvironment has recently been
suggested to influence HSC aging, but the underlying mechanisms remain largely unknown. Here we show that HSC aging criti-
cally depends on bone marrow innervation by the sympathetic nervous system (SNS), as loss of SNS nerves or adrenoreceptor
β​3 signaling in the bone marrow microenvironment of young mice led to premature HSC aging, as evidenced by appearance of
HSC phenotypes reminiscent of physiological aging. Strikingly, supplementation of a sympathomimetic acting selectively on
adrenoreceptor β​​3 to old mice significantly rejuvenated the in vivo function of aged HSCs, suggesting that the preservation or
restitution of bone marrow SNS innervation during aging may hold the potential for new HSC rejuvenation strategies.

M
ammalian aging can be described as a time-dependent
functional decline in the physiologic homeostasis of many
tissues, leading to increased risk of cardiovascular dis-
eases, neurodegenerative diseases, cancer, and diabetes1. One of
the major causes of age-associated tissue attrition is a functional
decline in tissue-specific stem cells2. In the hematopoietic system,
lifelong blood production depends on the ability of HSCs to self-
renew, differentiate, and form all blood cell lineages3. Aging of the
blood system is associated with myeloproliferation, immune senes-
cence, and anemia, attributed to age-dependent decline in HSC
function due to loss of regenerative potential and myeloid-biased
differentiation4. Studies have identified multiple HSC intrinsic fac-
tors that regulate their aging. Among these are mechanisms con-
trolling HSC metabolism (autophagy, mitochondrial dysfunction,
and nutrient sensing)5–9, replicative stress10, and DNA damage and
repair responses11–14. It has also been suggested that alterations in
the epigenetic landscape and cell polarity may drive HSC aging
manifestations14–16. Recent studies indicate that aging is also associ-
ated with drastic changes to the bone marrow microenvironment
and suggest that factors extrinsic to HSCs may promote their aged
phenotype17–21.
HSCs reside in a specialized microenvironment in the bone
marrow (also referred to as a niche), which represents a critical
regulatory unit essential to maintaining healthy hematopoiesis22.
HSC niches have recently been identified as perivascular units22
where subsets of quiescent HSCs are closely associated with arte-
riolar perivascular Nestin-GFP+ mesenchymal stem cells (MSCs),
glial fibrillary acidic protein (GFAP)-expressing Schwann cells
from adrenergic nerves, and megakaryocytes17,23–29. The sympa-
thetic nervous system (SNS) represents an important regulatory
component of the HSC niche, orchestrating release of ­adrenergic
neurotransmitter into the microenvironment in a circadian
­manner30–32. These autonomic signals regulate the proliferative
state of Nestin-GFP+ MSCs, HSC mobilization, and the hema-
topoietic regenerative capacity following genotoxic stress33–35.
Here we have evaluated the impact of aging on the bone marrow
microenvironment and have found, unexpectedly, that the loss of
sympathetic nerve fibers around arteriolar niches was a potent
driver of hematopoietic aging.
Results
Aging-related alterations of HSC niches. To define how aging
impacts HSC niches, we compared the bone marrow (BM) vascu-
lar architecture by whole-mount three-dimensional confocal fluo-
rescence imaging24 of young (8- to 10-week-old) and old (20- to
24-month-old) C57BL/6 mice and Nestin-GFP mice, in which GFP
marks putative HSC niche cells23. Nestin-GFP+ niche cells can be
divided into two distinct subpopulations based on GFP expression:
Nestin-GFPbright cells are exclusively found along arteries, while the
more abundant Nestin-GFPdim population is largely associated with
sinusoids24. Consistent with a recent study describing aging-related
alterations in BM17, we found that aging imposed drastic remodel-
ing of BM vascular architecture (Fig. 1a), as evidenced by an overall
increase in vascular density (Fig. 1b) and apparent deterioration of
arteriolar structures marked by significant shortening of Nestin-
GFPbright arteriole segments (Fig. 1c) and loss of α​-smooth muscle
actin-positive (α​-SMA+) cell density (Fig. 1d). FACS analyses con-
firmed the imaging results and revealed that the absolute number
of CD45−Ter119−CD31high total endothelial cells (ECs) were signifi-
cantly increased (P <​0.0001) while CD45−Ter119−CD31highSca-1high
arteriolar ECs were reduced (P <​0.0001) in aged mice compared to
young BM counterparts (Supplementary Fig. 1a).

1
Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Albert Einstein College of Medicine, New York, NY,
USA. 2Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York, NY, USA. 3Department of Medicine, Albert Einstein College
of Medicine, Bronx, New York, NY, USA. 4Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
5
Department of Medical Physics, Memorial Sloan Kettering Cancer Center, New York, NY, USA. 6Present address: Okayama University Hospital,
Department of Hematology and Oncology, Okayama, Japan. 7These authors contributed equally: Ali H. Zahalka, Halley Pierce.
*e-mail: paul.frenette@einstein.yu.edu

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Nature Medicine Articles
a Young Old

CD31 CD144 α-SMA Nes-GFPbright

b Vascular density c Arteriolar segment d α-SMA+ density e

Nes-GFPbright density
0.4 400 0.012 0.020
Ratio of CD31+CD144+

Ratio of Nes-GFPbright
P = 0.0016 P = 0.008 P = 0.0009
P = 0.0018

Ratio of α-SMA+
area/femur area

area/femur area

area/femur area
0.3 300 0.015
arteriole (μm)
Nes-GFPbright
0.008
0.2 200 0.010
0.004
0.1 100 0.005

0.0 0 0.000 0.000

Young Old Young Old Young Old Young Old

f Gated on live g
CD45– Ter119– CD31–
250,000
Young Old
Young Young
200,000 Nes-GFPbright 50
0.0046% 0.06 Old
150,000
Nes-GFPbright cells (%)

P = 0.02
100,000 0.05 40
Percent HSCs

P = 0.0047
50,000
0.04 30
1,000 CD31 CD144 CD150
3 3 4
–10 0 10 10 10
5
0.03 Lineage CD48 CD41
250,000 20
Old 0.02
200,000 Nes-GFPbright 10
150,000 0.044% 0.01
100,000 0.00 0
SSC-A

50,000 Young Old

0

0
00

20

40

60

80

00

20
–2

–4

–6

–8
–1

–1

–1

–1

–1

–2

–2
1,000
3 3 4 5
–10 0 10 10 10
Distance from arteriole (μm)
Nes-GFP

Fig. 1 | Aging induces remodeling of the HSC niche. a, Representative confocal z-stack projection montages of femurs from young (2 months) and old
(20–24 months) Nestin-GFP mice stained for double-positive CD31+CD144+ vasculature and α​-SMA+ cells with anti-CD31, anti-CD144, and anti-α​-SMA
antibodies. Scale bars, 500 µ​m for montages and 100 µ​m for zoomed projections; three independent experiments yielded similar results. b, Vascular
density in young and old mice, as assessed by quantification of CD31+CD144+ double-positive vascular area divided by total femur area (n =​9 and 17
projections in young and old mice, respectively; 4 mice per group). c, Arteriolar segment length in femurs of young and old Nestin-GFP mice, as assessed
by quantification of the length of the Nestin-GFPbright signal covering CD31+CD144+ double-positive arterioles (n =​11 and 6 projections in young and old
mice, respectively; 4 mice per group). d, α​-SMA+ cell density in young and old mice, as assessed by quantification of α​-SMA+ area divided by total femur
area (1 projection per mouse in young (n =​6) and old (n =​ 3) mice). e, Nestin-GFPbright density in young and old mice, as assessed by quantification of
Nestin-GFPbright area divided by total femur area (n =​9 and 17 projections in young and old mice, respectively; 4 mice per group) f, Left: representative
FACS plots of CD45−Ter119−CD31−Nestin-GFPbright MSCs isolated from young (top) and old (bottom) Nestin-GFP mice. SCC-A, side-scatter area. Gates and
percentages represent frequency of Nestin-GFPbright MSC population. Right: the frequency of Nestin-GFPbright MSCs from young and old Nestin-GFP mice
(n =​3 young and 4 old mice). g, Representative whole-mount projections (inset) and quantification of the distribution of Lin−CD41−CD48−CD150+
phenotypic HSCs in the sternal BM relative to Nestin-GFPbrightCD31+CD144+ double-positive arterioles in young and old C57BL/6 mice (n =​ 47 young
HSCs; 148 old HSCs; 3 mice per group; two-sample Kolmogorov–Smirnov test, P =​0.0047). Arrows denote HSCs. Scale bar, 100 µ​m. Data represented as
mean ±​ s.e.m.; P value determined by two-tailed t test unless otherwise indicated.

Imaging of the rare Nestin-GFPbright population revealed a sig- ­megakaryocytes24,26,27,36. To assess the relationship between HSCs
nificant expansion of these cells in old femurs (Fig. 1e), which with niche structures during aging, we imaged endogenous
was further confirmed by FACS analysis, demonstrating a specific CD150+CD48−CD41−Lin− HSCs in whole-mount sterna from
expansion of Nestin-GFPbright cells with age, without significant young and old mice. We found that HSCs in old mice were dis-
changes in the sinusoidal Nestin-GFPdim population (Fig. 1f and tributed significantly away from either niche structure compared
Supplementary Fig. 1b). Notably, expanded niche cells were distrib- to young animals (Fig. 1g and Supplementary Fig. 2a) and some-
uted away from endosteal region, converging significantly closer to times found in clusters, away from arterioles or megakaryocytes
the central vein (Supplementary Fig. 1c,d, P =​0.004). However, the (Fig. 1g). The loss of association with megakaryocytes was observed
frequency of Nestin-GFP+ MSCs from compact bones were signifi- despite their overall increased numbers in the BM of old mice
cantly reduced and had a lower colony-forming capacity in culture (Supplementary Fig. 2b,c), with increased numbers of megakaryo-
(Supplementary Fig. 1e,f; P =​0.03), indicating differential aging cyte progenitors and age-associated augmentation of platelet counts
manifestations between bone and marrow MSCs. (Supplementary Fig. 2d,e). These results indicate that aging induces
We and others have recently reported that subsets of qui- remodeling of HSC niches, associated with attrition of arteriolar
escent HSCs are significantly associated with arterioles and structures, expansion of total vascular density, and expansion of

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Articles
Nestin-GFPbright MSCs and megakaryocytes, leading to redistribu-
tion of old HSCs away from their putative niches.
Aging induces the loss of niche-associated adrenergic nerves. The
observed arteriolar attrition in old femoral BM (Fig. 1a–c and ref. 17)
suggested that aging may compromise SNS innervation, as these
nerves are tightly associated with arterioles22,24 and are reported
to be lost in hematological malignancies37,38. Staining for tyrosine
hydroxylase positive (TH+) adrenergic nerve fibers revealed a dra-
matic overall reduction in nerve density in old compared to young
femurs (Fig. 2a,b), as well as a significant loss of TH+ nerves per
Nestin-GFPbright arteriole (Fig. 2c). Staining of young and old femurs
with the pan-neuronal marker β​-III tubulin confirmed that aging
induced a dramatic loss of BM innervation (Fig. 2d,e). To evalu-
ate the possibility of neural dysfunction, we assessed the number
of synaptic contacts between nerves and blood vessels by staining
for synaptophysin39. In young mice, the staining pattern of synap-
tophysin exhibited a spiral-like pattern (similarly to TH staining;
Fig. 2c) and highly overlapped with β​-III tubulin staining. However,
upon aging, the number of synaptophysin+ synapses were signifi-
cantly reduced (Fig. 2f), suggesting a reduction in synapse density
per nerve. The loss of SNS was not generalized during aging because
the nerve density in the aging prostate, an organ highly innervated
by the SNS40, was not significantly altered compared to young
mice (Supplementary Fig. 3a; P =​0.285). Additionally, the expres-
sion of nerve growth factor (Ngf), a major neurotrophin for SNS
nerves41, which is selectively expressed by MSCs in BM, was sig-
nificantly reduced upon aging (Supplementary Fig. 3b; P =​ 0.004).
As SNS nerves regulate HSC egress in a circadian manner23,30,31,33,
we evaluated whether the age-associated BM SNS neuropathy had
a functional consequence for circadian rhythms of circulating pro-
genitors. Whereas the number of phenotypic (Lin−Sca-1+c-Kit+) or
functional (colony-forming units in culture) progenitors oscillated
in a circadian manner in the blood of young mice, the oscillations
of blood progenitors were ablated in old mice (Fig. 3a,b). These
changes were matched by inverse oscillations of HSC retention fac-
tor Cxcl12 mRNA levels in CD45−Ter119−CD31−CD51+PDGFRα​+
MSCs42 of young mice, while no significant circadian changes were
detected in old MSCs (Fig. 3c). Thus, these results suggest that the
aging HSC niche is associated with the profound loss of functional
SNS nerves.
Aging expands bone marrow MSCs with reduced HSC
maintenance capacity. Our previous studies have revealed
that CD45−Ter119−CD31−CD51+PDGFRα​+ cells highly over-
lap with the Nestin-GFP+ population and contain most
MSC- and HSC-maintaining activity42. Consistent with the
notions that SNS nerves negatively regulated BM MSC prolif-
eration35 and that aging caused BM SNS neuropathy, we found
that, similarly to Nestin-GFPbright stromal cells (Fig. 1e,f),
CD45−Ter119−CD31−CD51+PDGFRα​+ stromal cells obtained from
old mice were expanded (Fig. 3d and Supplementary Fig. 4b) and
were significantly more proliferative compared to young counter-
parts, as determined by Ki-67 expression (Fig. 3e). Although aging
significantly increased the overall BM cellularity (Supplementary
Fig. 4a; P <​0.0001), we did not detect any changes in the absolute
numbers and frequency of CD45−Ter119−CD31−CD51−PDGFRα​−
non-MSC cells (Supplementary Fig. 4c), suggesting specificity in
MSC expansion. MSCs derived from old mice exhibited lower clo-
nogenic potential in vitro (Fig. 3f,g), with reduced osteogenic poten-
tial (Supplementary Fig. 4d,e), and enhanced in vivo adipogenic
differentiation following sublethal irradiation, as determined
by perilipin+ areas (Supplementary Fig. 4f), in line with previ-
ous reports43. In addition, sorted MSCs from old mice expressed
lower mRNA levels of the canonical niche factors, Cxcl12, Scf, and
Angpt1 (Fig. 3h), suggesting reduced HSC-maintenance activity.
784
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These results thus indicate that the loss of SNS nerves may have
functional consequences on MSC function during niche aging.
To define better the onset of HSC niche aging, we evaluated
Nestin-GFP BM at 2, 8, and 12 months of age. These analyses revealed
that the loss of TH+ nerves (P =​0.0016) and reduced arteriolar seg-
ment length (P =​0.005), as well as the increased numbers of Nestin-
GFPbright cells (P =​0.02) or CD45−Ter119−CD31−CD51+PDGFRα​+
MSCs (P =​0.0015), were already apparent at 8 months of age, whereas
the changes in overall vascular density were not statistically sig-
nificant at this time (Supplementary Figs. 1a, 4b, and 5a–e). These
findings are consistent with a recent report documenting the emer-
gence of aging phenotypes, such as HSC expansion and lymphoid
dysfunction, from 8 months of age44. We also confirmed that the
frequency and absolute numbers of phenotypic HSCs (Lin−CD48−c-
Kit+Sca-1+CD150+) were significantly increased at 8 months of
age (P =​0.04), and found that the HSC population was signifi-
cantly redistributed away from arterioles at this age (Supplementary
Fig. 5f,g; P <​0.0001). Thus, SNS neuropathy in BM is an early hall-
mark of the aging HSC niche, apparent in 8-month-old adult mice
and concurrent with HSC aging.
Loss of SNS-nerves induces premature niche and HSC aging.
The loss of sympathetic innervation could merely accompany the
vascular remodeling and aging-associated alterations in hematopoi-
esis, or alternatively, it could represent a driver of the aging process.
To investigate this critical issue, we unilaterally denervated the hind
limb of young Nestin-GFP or C57BL/6 mice by surgical transection
of the femoral and sciatic nerves, as previously described31,45,46, while
the contralateral limb was sham-operated (Fig. 4a). This surgical
strategy, of nerve transection in the abdominal area close to their
origin in the spinal column and before neural branching, resulted
in a complete loss of TH+ nerves, specifically in the denervated
femur (Supplementary Fig. 6), and thus enabled us to compare the
impact of neural input on the aging phenotype in the same ani-
mal. As we have previously reported, acute BM denervation using
6-hydoxydopamine (6OHDA), 7 d post-denervation, did not result
in a detectable HSC phenotype at steady state35. Thus, to evaluate
whether SNS nerves play a role in the aging process, we denervated
mice in their youth (2 months old) and killed them for analysis at
4 months postsurgery (6 months of age), to avoid extending the age
over 8 months when aging phenotypes are apparent (Supplementary
Figs. 1a, 4a–c, and 5a–g and ref. 44).
Strikingly, we found that phenotypic HSCs were more pro-
liferative (Supplementary Fig. 7a) and expanded in the dener-
vated but not in the nerve-intact femurs (Fig. 4b), with specific
expansion of myeloid-biased CD41+ HSCs (Supplementary
Fig. 7b), a phenotype reminiscent of chronologically aged
HSCs47. Competitive repopulation assays using whole bone mar-
row (WBM) revealed increased overall donor-derived repopula-
tion activity in the denervated bone, compared to nerve-intact
bone, with myeloid-biased differentiation 4 months follow-
ing transplantation and increased engraftment of donor HSCs
(Supplementary Fig. 7c–i), which is also similar to the previously
reported competitive advantage of WBM transplantation from
old mice48,49. Using a separate cohort of mice, we also evaluated
the competitive repopulating capacity of 200 sorted HSCs from
denervated and nerve-intact BM to assess their self-renewal and
differentiation potential. These results revealed dramatic reduc-
tions of engraftment capacity and lymphoid cell production of
HSCs harvested from denervated (referred to as denervated
HSCs) compared to nerve-intact BM, which was maintained
upon secondary transplantation (Fig. 4c,d). To confirm further
that unilateral surgical denervation led to aging of HSCs, we
comparatively evaluated the polarity and DNA damage of dener-
vated and nerve-intact HSCs, as aging has previously been sug-
gested to be associated with a loss of polarity and increased DNA
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Nature Medicine Articles
a TH+ adrenergic innervation
Young Old

Nes-GFPbright
CD31 CD144
TH

b TH per femur c TH per arteriole

2.0 Young 3.0 P = 0.004
Ratio of TH+ area/femur
area (relative to young)

P = 0.0001
2.5

Ratio of TH+ area/

Nes-GFPbright area
1.5
2.0
1.0 1.5
Old
1.0
0.5
0.5
0.0 0.0
Young Old TH CD31 CD144 Nes-GFPbright Young Old

d Total neural innervation

Young Old

CD31 CD144 β-III tubulin

e f Young Old
Total nerve density Synapse density
Ratio of synaptophysin

0.005 3.0
area/β-III tubulinarea

P = 0.0016
Ratio of β-III tubulin

2.5 P = 0.0002
area/femur area

0.004
2.0
0.003
1.5
0.002
Merge Merge 1.0
0.001 0.5
0.000 0.0
Young Old CD31 CD144 β-III tubulin Synaptophysin Young Old

Fig. 2 | Aging induces the loss of niche-associated adrenergic nerves. a, Representative confocal z-stack projection montages from the BM of young
and old Nestin-GFP mice stained for CD31+CD144+ double-positive vasculature and TH+ nerve fibers. Scale bar, 500 µ​m. Arrows denote TH+ nerves.
b, Femoral adrenergic innervation quantified by TH+ area divided by total femur area (normalized to young; n =​7 and 12 old projections in young and
old mice, respectively; 4 mice per group). c, Left: representative confocal z-stack projections from the BM of young and old Nestin-GFP mice stained
for CD31+CD144+ double-positive vasculature and TH+ nerve fibers. Scale bar, 100 µ​m. Right: arteriolar adrenergic innervation quantified by TH+ area
divided by total Nestin-GFPbright arteriole area (normalized to young; n =​4 and 10 projections in young and old mice, respectively; 4 mice per group).
d, Representative confocal z-stack projection montages from the femurs of young and old C57BL/6 mice stained for CD31+CD144+ double-positive
vasculature and β​-III tubulin+ nerve fibers. Scale bar, 500 µ​m. Arrows denote β​-III tubulin+ nerves. e, Total femoral innervation quantified by β​-III
tubulin+ area divided by total femur area (n =​9 and 7 projections in young and old mice, respectively; 3 mice per group). f, Left: representative confocal
z-stack projections from femurs of young and old C57BL/6 mice stained for CD31+CD144+ double-positive vasculature, β​-III tubulin+ nerve fibers and
synaptophysin+ (Syn+) synaptic vesicles. Scale bar, 100 µ​m. Right: synapse density as assessed by quantification of Syn+ area per β​-III tubulin+ nerve area
(n =​54 and 27 projections in young and old mice, respectively; 3 mice per group). Data represented as mean ±​ s.e.m.; P value determined by two-tailed
t test. At least three independent experiments yielded similar results presented in a and d. For box plots, the box spans from the 25th to 75th percentiles
and the centerline is plotted at the median. Whiskers represent minimum to maximum range.

damage in HSCs10,16. Indeed, immunofluorescence analysis of sorted foci formation was significantly increased in denervated HSCs
HSCs from young, old, sham, or denervated femurs revealed that compared to sham control, reaching frequencies similar to
Cdc42 and tubulin polarity was significantly altered, and γ​H2AX those seen in old HSCs (Fig. 4e,f and Supplementary Fig. 8a–c).

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Articles Nature Medicine

a Circulating CFU-C
b Circulating LSK
c MSC Cxcl12
2.5 4 2.5
ZT5 ZT5 ZT5

(normalized to young ZT5)

P = 0.454 ZT13

LSK per mL blood (fold)

ZT13

(relative to young ZT5)

2.0 P = 0.102 ZT13 2.0

CFU-C per mL blood

P = 0.426
3
P ≤ 0.0001

Relative mRNA
P ≤ 0.0001
1.5 1.5
2 P = 0.005
1.0 1.0

1
0.5 0.5

0.0 0 0.0
Young Old Young Old Young Old

d e CD45– Ter119–CD31–
Gated on live
CD51+PDGFRα+
CD45– Ter119–CD31–
CD45– Ter119–CD31– 105 Young
105 Young CD51+PDGFRα+ MSCs
MSCs 104 MSC G0
104
14 0.10 P = 0.002 3
100 P = 0.005
P = 0.006 10
103
12
MSCs per femur (×103)

0.023% 0.08 0 80
0 10 –103 G0 70.6%
MSCs (%)

Cells (%)
8 0.06 60

50 0
10 000
15 000
20 000

0, 0
0
0 103 104 105

25 ,00
00
,
0,
0,
0
6 0.04 40
105 Old
4 MSCs 4 5 Old
10 10
PDGFRα-APC

0.02 20
2
10 3 104
0 0.00 0

Ki-67-PerCP
0.068%
0 Young Old Young Old 103 Young Old
0
0 103 104 105 –103 G0 54.2%
CD51-PE

50 0
10 000
15 000
20 000

0, 0
0
25 ,00
00
,
0,
0,
0
Hoechst
33342
f g h HSC maintenance factors
Mesenspheres
CFU-F 1.5 Cxcl12 1.5 Scf 2.0 Angpt1
(as % of CD51+PDGFRα+ cells)
(as % of CD51+PDGFRα+ cells)

6 2.0
P = 0.0003 P ≤ 0.0001 P = 0.01
P ≤ 0.0001 P ≤ 0.0001
Relative mRNA (fold)

1.5
1.5 1.0
Mesenspheres

1.0
4
CFU-F

1.0 1.0

2 0.5 0.5
0.5 0.5

0 0.0 0.0 0.0 0.0

Young Old Young Old Young Old Young Old Young Old

Fig. 3 | Aging expands MSCs and reduces their HSC maintenance activity. a, b, Circadian oscillations of (a) circulating colony-forming units in culture
(CFU-C; normalized to young at Zeitgeber time (ZT) 5 (n =​13 young ZT5, 7 old ZT5, 7 young ZT13, and 4 old ZT13 mice) and (b) Lin−Sca-1+c-Kit+ (LSK)
progenitors (normalized to young at ZT5; n =​5 mice per group) in peripheral blood of young and old C57BL/6 mice. c, Quantification of Cxcl12 mRNA
levels relative to Actb in sorted MSCs from young and old C57BL/6 mice at ZT5 and ZT13 (normalized to young at ZT5; n =​8 young ZT5, 5 old ZT5,
5 young ZT13, and 4 old ZT13 mice). d, Left: representative FACS plots showing the gating strategy for CD45−Ter119−CD31−CD51+PDGFRα​+ MSCs in young
(top) and old (bottom) C57BL/6 mice. Gates and percentages represent frequency of CD51+PDGFRα​+ MSCs. Right: absolute numbers and frequency of
MSCs in young and old C57BL/6 mice (n =​4 mice per group). e, Left: representative FACS plots showing the gating strategy for MSCs Ki-67 and Hoechst
33342 staining in young (top) and old (bottom) C57BL/6 mice. Right: quantification of Ki-67− G0 MSCs in young and old C57BL/6 mice (n =​6 mice per
group). f,g, Frequency of (f) fibroblast colony-forming units (CFU-F; n =​15 cultures per group) and (g) mesenspheres (n =​9 young, 11 old cultures) from
sorted MSCs plated at equal numbers and clonal densities under CFU-F or mesensphere culture conditions (n =​5 mice per group). h, Quantification
of mRNA levels of Cxcl12, Scf, and Angpt1 relative to Gapdh in sorted MSCs (normalized to young; n =​7 young, 11 old mice). Data represented as
mean ±​ s.e.m.; P value determined by two-tailed t test.

These data suggest that the denervation leads to a premature seen in aged mice, characterized by arteriolar segment shorten-
aging-like HSC phenotype, with expansion of myeloid-biased ing (Fig. 4g) and loss of α​-SMA+ cells (Supplementary Fig. 8d).
HSCs with reduced long-term self-renewal capacity. In addition, surgical BM denervation in young mice led to aug-
To determine the impact of denervation on aging of the mentations in BM cellularity (Supplementary Fig. 8e) and in
HSC niche, we evaluated the vascular structures and their numbers of CD31high total ECs (Supplementary Fig. 8f) and
associated stromal cells by whole-mount immunofluores- CD45−Ter119−CD31−CD51+PDGFRα​+ MSCs (Fig. 4h), which
cence imaging and FACS. Remarkably, denervation induced a exhibited reduced clonogenic potential (Fig. 4i), as observed
remodeling of the BM microenvironment reminiscent of that in chronologically aged mice. Moreover, bone marrow MSCs

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Nature Medicine Articles
a b HSCs c Total blood Blood myeloid Blood B cells
60 P = 0.022 80 P = 0.03 60
6 P = 0.0021 P = 0.016
Sham Den P = 0.049
P = 0.02

HSCs per femur (×103)

Donor chimerism (%)

P = 0.024
60
4 40 40
P = 0.06
P = 0.026 40 P = 0.019
2 months
old 2 20 20
20
4 months
0 0 0 0
Analysis at 6 months old
Sham Den 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4
Den Sham Time (months)
d Primary Secondary e Sham
100 BM Sham 100 Den
P = 0.03

My
Donor chimerism (%)

80
P = 0.0001 P = 0.009

Polarized HSCs (%)

B 80
*
P = 0.014

T
60 60
P = 0.610 Den
40 P = 0.025 40
*
20 ** 20

0 Cdc42 Tubulin DAPI Merge 0

Sham Den Sham Den Cdc42 Tubulin
f g Sham Den
Young Old 100

DAPI
Arteriolar segment
P = 0.006
P ≤ 0.0001 400
80

Nes-GFPbright arteriole (μm)

P ≤ 0.0001
HSCs with γH2AX

CD31 CD144
60 300
foci (%)

Sham Den 40 200

20 100

Nes-GFPbright
0
0
γH2AX DAPI Young Sham
Sham Den
Old Den

h Gated on live i j
CD45–Ter119–CD31– MSCs CFU-F Cxcl12 Scf Angpt1 Vcam1
Sham 3 1.5 P ≤ 0.0001 P = 0.004 P = 0.024 P = 0.037
30 P = 0.0039 P = 0.013
MSCs
CD51+PDGFRα+ cells)
MSCs per femur (×103)

Relative mRNA (fold)

CFU-F (as % of

20 2 1.0
0.06%
PDGFRα-PE/Cy7

Den 10 1 0.5
MSCs

0 0 0.0
0.08%
Sham Den Sham Den Sh D Sh D Sh D Sh D

CD51-PE

Fig. 4 | Surgical denervation of young BM induces premature HSC and niche aging. a, Schematic illustration of the surgical denervation experiment. The
sciatic and femoral nerves were transected in young (2-month-old) C57BL/6 mice and the mice were analyzed 4 months postsurgery, at 6 months of age.
b, Absolute numbers of HSCs (Lin−Sca-1+c-Kit+CD48–CD150+) isolated from sham and denervated (Den) femurs (n =​ 7 mice). c, Total peripheral blood
(CD45.2+), blood myeloid (Mac-1+CD45.2+), and blood B cell (B220+CD45.2+) donor chimerism at the indicated timepoints post-transplantation. d, Bone
marrow donor chimerism (total CD45.2+ (BM); myeloid (My), Mac-1+CD45.2+; B cell (B), B220+CD45.2+; and T cell (T), CD4+CD8+CD45.2+) 5 months
after (left) primary transplantation of 200 HSCs derived from either sham or Den femurs and transplanted in competition with young BM competitor cells
(n =​4 sham, 6 denervated mice) and (right) 5 months after secondary transplantation of 3 ×​ 106 BM from primary recipients. e, Left: representative confocal
z-stack projections of HSCs sorted from sham or denervated femurs and stained with Cdc42, tubulin, and DAPI. Scale bar, 10 µ​m. Right: quantification of the
percentage of Cdc42 and tubulin polarized HSCs out of total HSCs scored (total of 356 sham and 353 denervated HSCs isolated from 4 mice per group).
f, Left: representative confocal z-stack projections of HSCs sorted from young, old, sham, or denervated femurs and stained with γ​H2AX and DAPI. Scale
bar, 10 µ​m. Right: quantification of the percentage of HSCs with γ​H2AX foci (calculated as the mean percentage of a total of 164 young, 247 old, 381
sham, and 355 Den HSCs isolated from 3 young, 3 old, and 4 denervated mice). g, Left: confocal z-stack projection montages from the femurs of sham
and denervated tibiae from Nestin-GFP mice stained for CD31+CD144+ double-positive vasculature and DAPI. Scale bar, 500 µ​m. Right: assessment of
arteriolar segment length as assessed by quantification of the length of the Nestin-GFPbright signal covering CD31+CD144+ double-positive arterioles in sham
and denervated tibiae (n =​8 and 11 sham and denervated projections, respectively; 4 mice). h, Left: representative gating strategy for FACS quantification
and sorting of MSCs (CD45−Ter119−CD31−CD51+PDGFRα​+) in sham (top) and denervated (bottom) femurs. Gated population and percentages represent
frequency of CD51+PDGFRα​+ MSCs. Right: quantification of MSCs isolated from sham and denervated femurs (n =​ 6 mice). i, Frequency of CFU-F from
sorted MSCs from denervated femurs, plated at equal numbers at clonal densities under CFU-F culture conditions (n =​ 4 mice). j, Quantification of mRNA
levels of Cxcl12, Scf, Angpt1, and Vcam1 relative to Gapdh in sorted bone marrow MSCs derived from sham (Sh) and denervated (D) tibia (normalized to
sham; n =​6 mice). Data represented as mean ±​ s.e.m.; P values determined by two-tailed paired t test in b, h–j and by two-tailed unpaired t test in c–g.

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Articles
harvested from denervated bones exhibited reduced expression of
major HSC-maintenance factors (Fig. 4j) and diminished osteo-
genic potential (Supplementary Fig. 8g), compared to those har-
vested from nerve-intact contralateral bones. These data strongly
indicate that the premature loss of SNS nerves in young mice can
drive the emergence of an aged HSC niche.
Because unilateral surgical denervation results in complete
immobility of the denervated limb, it could be argued that the
aging-like HSC and niche phenotypes could be an indirect effect of
reduced limb mobility on the skeleton or to collateral denervation
of nonhematopoietic organs (for example, muscle). To evaluate this
possibility, we performed a musculocutaneous denervation of the
hindlimb by transecting the sciatic nerve distal to the spinal cord
close to the sciatic notch and the femoral nerve distal to the spinal
cord, as previously reported not to directly affect bone and marrow
homeostasis50,51. As expected, hindlimb musculocutaneous dener-
vation did not eliminate sympathetic nerves in denervated femurs
and did not induce any aging-like HSC and niche phenotypes
4 months after surgery (Supplementary Fig. 9a–c), confirming that
aging-like phenotypes observed in denervated femurs are directly
related to BM denervation.
HSC aging depends on niche-derived ADRβ3 signals. SNS
nerves locally deliver noradrenaline as its major neurotransmitter
targeting β​2 (ADRβ​2) and β​3 (ADRβ​3) adrenergic receptors in the
BM microenvironment31,33,34. While Adrb2 is broadly expressed in
both hematopoietic and nonhematopoietic compartments, Adrb3
expression is restricted to the stroma and does not change with aging
(see our analyses of published HSC52 and stromal53 RNAseq data-
sets, as well as Supplementary Fig. 10a–c). We thus tested whether
the administration of two sympathomimetics, an ADRβ​2-selective
agonist (clenbuterol) and an ADRβ​3-selective agonist (BRL37344),
could rejuvenate the HSC niche and HSCs of old mice. Continuous
sympathomimetic delivery was achieved by serial subcutaneous
implantations of Alzet osmotic pumps in aged C57BL/6 animals
to achieve drug delivery over 12 weeks. As controls, both young
and old mice received pumps filled with saline (Supplementary
Fig. 11a). Treatment of old mice with either sympathomimetic
was well tolerated and did not produce any hematological toxicity
as determined by stable blood counts at the termination of drug
delivery (Supplementary Table 1). Remarkably, the administra-
tion of either sympathomimetic lowered the absolute numbers and
frequencies of MSCs and ECs to levels similar to those observed
in young mice, without detectable alterations in BM cellularity
(Fig. 5a and Supplementary Fig. 11b–e), suggesting a potential for
niche rejuvenation. Additionally, treatment of old mice with either
β​-adrenergic-selective sympathomimetic significantly reduced the
number of phenotypic HSCs (Fig. 5b and Supplementary Fig. 11f).
To test the rejuvenation potential of these agents on HSC func-
tion, we then competitively transplanted sorted HSCs obtained
from these treated mice. While ADRβ​2 agonist treatment did not
improve old HSC engraftment or contribution to the lymphoid lin-
eage, treatment with the ADRβ​3 agonist markedly increased donor
chimerism to near-normal levels in peripheral blood and BM 5
months following transplantation (Fig. 5c). In addition, the ADRβ​
3 agonist significantly improved multilineage cell production and
HSC engraftment (Fig. 5c and Supplementary Fig. 11g–j). The
functional rejuvenation of old HSCs by ADRβ​3 agonist treatment
was also evident following secondary BM transplantation, in which
the enhanced BM multilineage contribution and HSC engraftment
persisted (Fig. 5d). A separate cohort of animals competitively
transplanted with WBM also showed similar signs of rejuvena-
tion only in the ADRβ​3 agonist-treated group, characterized by
a reduction in the competitive advantage of old WBM and HSC
expansion, indistinguishable from that of saline-treated young
mice (Supplementary Fig. 12a,b). We also administered the ADRβ​
788
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Nature Medicine
3 agonist BRL37344 to denervated mice and found that it could res-
cue the premature aging phenotypes (Supplementary Fig. 13a–f),
suggesting specificity in the effect of ADRβ​3 signaling in the BM
microenvironment.
To confirm further that ADRβ​3 agonist treatment rejuvenated
old HSCs and to obtain further molecular insight, we analyzed
the transcriptomes by RNA-seq of HSCs sorted from young mice
(yHSCs), old mice (oHSCs), or ADRβ​3-agonist rejuvenated old
mice (rHSCs). Using t-distributed stochastic neighbor embedding
analysis54, we found that the rHSC cluster did not overlap with
oHSCs and moved closer to yHSCs (Supplementary Fig. 14a), sug-
gesting that ADRβ​3 agonist treatment altered the gene expression
patterns of old HSCs. Unsupervised hierarchical clustering of top
differentially expressed genes also revealed that the rHSCs’ expres-
sion signature became similar to that of yHSCs (Supplementary
Fig. 14b). Pathway analysis of top differentially expressed genes
between rHSCs and oHSCs revealed downregulation of genes asso-
ciated with active cell cycle progression, primarily mitosis and cyto-
kinesis (Supplementary Fig. 14c), suggesting that treatment with the
ADRβ​3 agonist impacted cell cycle status of old HSCs, reported to
be highly variable and a major contributor of heterogeneity within
the aged HSC pool10,55. Moreover, upregulation of genes associated
with chemokine activity and cytoskeleton organization was detected
in rHSCs (Supplementary Fig. 14c), pathways reported to be dimin-
ished in old HSCs, and contributing to enhanced mobilization and
poor homing activity56–58. Further evaluation of lineage-selective
gene sets, focusing on those reported to be differentially expressed
in old HSCs11, revealed a restoration toward levels in yHSCs, with
more profound effects observed for the lymphoid-lineage genes
(Fig. 5e). These data indicate that ADRβ​3 reprograms an array of
pathways that confer rejuvenated phenotypic characteristics.
The rejuvenation of old HSCs was likely mediated by the niche
because, in contrast to Adrb2, which is expressed by HSC and pro-
genitors, neither young nor old HSCs expressed Adrb3 transcript
or responded to ADRβ​3 agonist BRL37344 in vitro (Supplementary
Fig. 10a,b). In keeping with an action of the ADRβ​3 agonist on the
niche, immunofluorescence imaging of vascular structures and their
associated stromal cells revealed partial restoration of arteriolar seg-
ment length and α​-SMA+ cell density by ADRβ​3 agonist treatment
(Supplementary Fig. 14d–f). In addition, a more detailed analysis
of CD45−Ter119−CD31−CD51+PDGFRα​+ MSC functional proper-
ties revealed significant improvements in Cxcl12, Scf niche factors,
Ngf expression (Fig. 5f), and CFU-F (fibroblast colony-forming
unit) activity (Fig. 5g), suggesting a rejuvenated state of aged MSCs.
These results suggest that niche rejuvenation by supplementation of
ADRβ​3 signals in old mice significantly rejuvenates old HSCs and
restored their functional properties.
Deletion of ADRβ3 in mice induces an accelerated aging of
HSC niches. Since ADRβ​3 mimetics rejuvenated the HSC niche
in old mice, we tested whether the constitutive deletion of Adrb3
(Adrb3−/−) could lead to premature aging. Serial analyses of
Adrb3−/− and control mice at 2.5 and 5 months of age showed a
significant expansion of MSCs with reduced CFU-F and HSC
maintenance activities at 2.5 months (Fig. 5h–j), without detectable
alterations in BM cellularity or CD31high total ECs (Supplementary
Fig. 15a,b). In addition, while other aging phenotypes such as
reduction in Sca-1highCD31high arteriolar ECs (Supplementary
Fig. 15c) and HSC expansion (Fig. 5k) were not present at 2.5
months of age, they were apparent at 5 months, suggesting that
loss of ADRβ​3 signaling in stromal cells may be an initial event
leading to premature HSC aging. It is notable that the expanded
HSCs were significantly enriched in CD41+ myeloid-biased HSCs47
(Supplementary Fig. 15d; P =​0.03), with reductions in lymphoid-
primed multipotent progenitors (Fig. 5l), common lymphoid pro-
genitors (Supplementary Fig. 15e), and a trend toward increased
Nature Medicine | VOL 24 | JUNE 2018 | 782–791 | www.nature.com/naturemedicine
Nature Medicine Articles
a b c Primary HSC transplantation (5 months)
MSCs HSCs Total blood Myeloid Lymphoid HSCs
P = 0.0018
30 P = 0.0001 30 120 P = 0.006

MSCs per femur (×103)

HSCs per femur (×103)

P ≤ 0.0001

Donor chimerism (%)

P = 0.004
P ≤ 0.0001 P ≤ 0.0001 100 P = 0.05
P ≤ 0.0001 P = 0.007
20 20 80
P = 0.004
60 P = 0.0021
P = 0.06
10 10 40
P = 0.05
20
0 0 0

d Secondary transplantation (5 months) e

Myeloid Lymphoid Mk/platelet
Total BM Myeloid Lymphoid HSCs 1.0
120
Rab27b Stra13 Vwf
Donor chimerism (%)

P = 0.0009 0.5

z-score
100 P = 0.002 P ≤ 0.0001 Fgfr1op2 Tia1 Clu
P = 0.007 Hoxb6 Blnk Sdpr 0
80 P = 0.0002 P = 0.02 Hoxb8 Csk Itgb3
P = 0.0016 Osmr Dntt Fhl1
–0.5
60 Plscr2 Elmo1 Tgm2 –1.0
P = 0.043 Anxa7 Flt3
40 Fgr Sdc1 Lymphoid genes Myeloid genes
Fli1 Il15
NES = 1.51
Il17ra
20 Gp1bb
Irf2
P = 0.00
FDR = 0.11
Hip1 NES = –1.34
Lgals3
0 Fgfr1
Mtcp1
P = 0.11
FDR = 0.15
Trim24
f 2.0 P = 0.0198
Epb41 Pik3ap1
Pou2af1
‘YS’ (positively correlated)

yHSC
‘YS’ (positively correlated)

yHSC
Dyrk3
Relative mRNA (fold)

P = 0.02 Cebpb Satb1 Zero cross at 5938

oHSC
Zero cross at 5938

oHSC
P =0.042 Slamf6
P = 0.039
‘OS’ (negatively correlated) ‘OS’ (negatively correlated)

Cebpd
1.5 Mkl1
Sox4 NES = 1.48
P = 0.0065 Ebi3 P = 0.00
P = 0.042 Pml FDR = 0.10
Tnfrsf13c NES = –0.72
Ucp2 P = 0.81
1.0 Ptpn11
Wasl FDR = 0.79
Bcl3
Ncf4
Ddx23
Whsc1l1 ‘OB’ (positively correlated) ‘OB’ (positively correlated)

0.5 Camp
Ikbkb rHSC Zero cross at 5898
rHSC Zero cross at 5898

Rnf128 oHSC oHSC

Mpo Ezh2
Cxcl12 Scf Ngf
‘OS’ (negatively correlated) ‘OS’ (negatively correlated)

Ngp
0.0 S100a8
Fyb
yHSC (saline) oHSC (saline)
Hcst
S100a9
Y-saline O-saline O-clenbuterol O-BRL37344 Erg
rHSC (BRL37344)

g h Adrb3 +/+ Adrb3 –/–

i j
2.0 Adrb3 +/+
1.5
(normalized to Adrb3 +/+)

4 P = 0.047 2.0 Adrb3 –/– P = 0.02

Relative mRNA (fold)
P = 0.0009
P = 0.0132
MSCs per femur

1.5 P = 0.011
CFU-F (fold)

CFU-F (fold)

P = 0.007 3 1.5 P = 0.048

P = 0.039 P = 0.002
1.0
1.0 2 1.0
0.5
0.5 1 0.5

0.0 0 0.0 0.0

2.5 5 Adrb3 +/+ –/– Cxcl12 Scf Angpt1 Ngf
Age (months)

k Adrb3 +/+ l Adrb3 +/+ Adrb3 –/– m n

8 Adrb3 –/– 15 40 P = 0.005
LMPPs per femur (×103)

100
HSCs per femur (×103)

P = 0.002 P = 0.76
PB lymphoid cells (%)
PB myeloid cells (%)

P = 0.001 P = 0.0002
P = 0.024 Adrb3 +/+ 80
6 30 Adrb3 –/–
10
60
4 P = 0.94 20
P = 0.25 40 Adrb3 +/+
5
2 10 20 Adrb3 –/–

0 0 0 0
2.5 5 2.5 5 2.5 5 2.5 5
Age (months) Age (months) Age (months) Age (months)

Fig. 5 | ADRβ3 signaling is essential for maintenance of aging HSCs. a,b, Absolute numbers of (a) MSCs (CD45−Ter119−CD31−CD51+PDGFRα​+) and (b)
HSCs (Lin−Sca-1+c-Kit+CD48−CD150+) in young and old mice implanted with osmotic pumps containing saline, the β​2-adrenergic agonist clenbuterol, or
the β​3-adrenergic agonist BRL37344 (n =​ 7 young +​saline (Y-saline), 6 old +​saline (O-saline), 8 old +​clenbuterol (O-clenbuterol), and 5 old +​ BRL37344
(O-BRL37344) mice). Colors as in f. c, Total blood chimerism (CD45.2+), blood myeloid chimerism (Mac-1+CD45.2+), blood lymphoid chimerism
(B220+CD4+CD8+CD45.2+), and HSC chimerism (Lin−Sca-1+c-Kit+CD48−CD150+CD45.2+) in CD45.1 recipient mice transplanted with 200 HSCs, derived
from the mice described in a and b, at 5 months after transplantation (n =​ 5 young +​saline, 4 old +​saline, 4 old +​clenbuterol, and 4 old +​ BRL37344 mice).
d, Bone marrow chimerism following secondary BM transplantation from the mice described in c, 5 months after transplantation. (n =​ 5 young +​ saline,
4 old +​saline, 5 old +​clenbuterol, and 5 old +​ BRL37344 mice). e, Heat map of mean gene expression levels in yHSCs, oHSCs, and rHSCs (n =​ 3 samples
per group) of selected myeloid, lymphoid, and megakaryocyte and platelet (Mk/platelet) lineage genes and signature enrichment plots from GSEA using
myeloid and lymphoid signature gene sets11. Shown are the normalized enrichment score (NES), P value, and false discovery rate (FDR) of the enrichment.
f, Quantification of mRNA levels of Cxcl12, Scf, and Ngf relative to Actb in sorted MSCs derived from mice described in a and b (n =​ 5 young +​ saline,
4 old +​saline, 5 old +​clenbuterol, and 4 old +​BRL37344; normalized to young +​ saline). g, CFU-F frequency from sorted MSCs derived from mice
described in a and b (normalized to young +​ saline; n =​ 5 young +​saline, 6 old +​saline, and 4 old +​ BRL37344 mice). h, Absolute numbers of MSCs from
Adrb3+/+ and Adrb3−/− mice at 2.5 and 5 months of age (normalized to Adrb3+/+; n =​4 mice per 2.5-month group, 6 Adrb3+/+, 7 Adrb3−/− per 5-month
group). i,j, CFU-F frequency from sorted MSCs (i; normalized to Adrb3+/+; n =​5 mice per group) and quantification of mRNA levels of Cxcl12, Scf, Angpt1,
and Ngf relative to Gapdh in sorted MSCs (j; normalized to Adrb3+/+; n =​ 5 Adrb3+/+ and 4 Adrb3−/− mice) from 2.5-month-old Adrb3+/+ and Adrb3−/− mice.
k–n, Absolute numbers of (k) HSCs, (l) lymphoid-primed multipotent progenitors (LMPPs; Lin–Sca-1+c-Kit+CD34+Flt3+), (m) peripheral blood (PB)
myeloid cells (Mac-1+Gr-1+), and (n) lymphoid cells (B220+CD4+CD8+) cells in Adrb3+/+ and Adrb3−/− mice at 2.5 and 5 months of age (n =​4 mice per
2.5-month group, 7 (k–i) and 8 (m,n) per 5-month group). Data represented as mean ±​ s.e.m.; P values determined by two-tailed t test. For box plots, the
box spans from the 25th to 75th percentiles and the centerline is plotted at the median. Whiskers represent minimum to maximum range.

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Articles
common myeloid progenitors and granulocyte-macrophage pro-
genitors (Supplementary Fig. 15f,g), leading to myeloid bias and
lymphopenia in peripheral blood (Fig. 5m,n), reminiscent of
premature aging. To confirm further that the accelerated aging
­phenotype observed in Adrb3−/− mice derives from the deletion of
Adrb3 in the niche, we carried out reciprocal BM transplantation of
young wild-type BM into lethally irradiated control (Adrb3+/+) or
Adrb3−/− young mice (Supplementary Fig. 15h). To detect prema-
ture aging phenotypes, chimeric mice were analyzed at 5 months
of age (3 months post-transplantation; Supplementary Fig. 15i).
We found that Adrb3 deletion in the microenvironment was suffi-
cient to induce aging-like changes in BM progenitors, although the
numbers of HSCs and lymphoid-primed multipotent progenitors
were not altered at this time (Supplementary Fig. 15k). Moreover,
we detected significant MSC expansion (P =​0.05) and evidence
of myeloid expansion with lymphopenia in the Adrb3-deficient
niche (Supplementary Fig. 15l–n). Thus, the loss of niche-derived
ADRβ​3 signaling in young mice promotes premature HSC aging.
Discussion
Here we show that aging induces significant remodeling of the
BM vasculature and its associated stromal cells with progres-
sive loss of SNS innervation. The SNS neuropathy of aging is not
a consequence of vascular attrition, but rather an early driver of
niche aging, as nerve loss in a model of surgical denervation or
genetic deletion of Adrb3 can induce dramatic remodeling of the
HSC niche. Our results show that the denervation-associated niche
remodeling leads to premature aging-like changes in HSCs. The
reprogramming of HSCs residing in denervated niches presents
all the hallmarks of aging, such as poor BM repopulating activity,
myeloid bias, and the acquisition of polarity defects with increased
frequency of γ​H2AX foci10,12. Notably, we show that the administra-
tion of an ADRβ​3 agonist can reverse, at least in part, the molecular
and functional aging signatures.
Extrinsic factors within the HSC niche have been suggested to
contribute to HSC aging. A recent study has found that aging-asso-
ciated alterations of arterioles was dependent on Notch signaling
and that, although overexpression of Notch could rescue the vas-
cular phenotype, it could not rejuvenate aged HSCs17, indicating
that HSC aging may depend on a more complex interplay among
niche components. Niche-derived soluble factors, including the
pro-inflammatory CC-chemokine ligand 5 (CCL5), osteopontin,
and CXCL12, have been reported to influence HSC aging18,20,21. Our
results show that the prolonged loss of ADRβ​3 signaling derived
from SNS nerves leads to a remodeling of HSC niches that promotes
premature aging.
While several intrinsic mechanisms have been proposed to
rejuvenate old HSCs4, the present results highlight key functions
of the niche in both aging and rejuvenation of HSC functions.
An extrinsic mechanism for BRL37344-mediated rejuvenation
is supported by the absence of Adrb3 expression in HSCs, the
lack of direct HSC response to BRL37344, and the reciprocal
transplantation of wild-type BM into Adrb3−/− animals, show-
ing that the deficit in ADRβ​3 signaling in the microenvironment
mediated the premature HSC aging. Given the broad expres-
sion changes in rejuvenated HSCs, it is likely that the molecu-
lar mechanism(s) of ADRβ​3 agonist is (or are) complex and may
involve multiple pathways emerging from the rejuvenated micro-
environment that remain to be defined. In this context, it is note-
worthy that sympathetic neural dysfunction has been described
in primary and secondary lymphoid organs, where remodeling
of sympathetic innervation in the spleen and thymus caused age-
related alterations in the local microenvironment and immune
dysfunctions59. Our results indicate that local BM neural degen-
eration may serve as a key driver of aging and provide the proof-
of-principle that rejuvenation of old HSCs can be achieved by
790
© 2018 Nature America Inc., part of Springer Nature. All rights reserved.
Nature Medicine
modulating the ­neural input to the niche. Protection of endoge-
nous SNS nerves or supplementation of ADRβ​3 signals in old age
may thus offer an exciting new avenue for niche-targeted stem
cell ­rejuvenation therapy.
Methods
Methods, including statements of data availability and any asso-
ciated accession codes and references, are available at https://doi.
org/10.1038/s41591-018-0030-x.
Received: 12 June 2017; Accepted: 12 March 2018;
Published online: 7 May 2018
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Acknowledgements
We thank J. Vijg (Department of Genetics, Albert Einstein College of Medicine)
for advice on experiment design and for providing old C57BL/6 mice for initial
experiments. We also thank C. Prophete for technical assistance, M. Lee for assistance
with old mice, and D. Sun and L. Tesfa for assistance with cell sorting and S. Maqbool
for RNA sequencing. We are also grateful to the New York State Department of Health
(NYSTEM Program) for shared facility (C029154) and research support (N13G-262)
and the Leukemia and Lymphoma Society’s Translational Research Program. This
work was supported by R01 or U01 grants from the National Institutes of Health (NIH)
(DK112976, DK056638, HL116340, HL097819, and DK116312 to P.S.F.) and by the
New York Stem Cell Foundation (NYSCF). M.M. is a New York Stem Cell Foundation
(NYSCF) Druckenmiller fellow and was previously supported by the EMBO European
Commission FP7 (Marie Curie Actions; EMBOCOFUND2012, GA-2012-600394, ALTF
447-2014). A.H.Z was supported by NIH Training Grant (T32 NS007098) and by a
National Cancer Institute (NCI) predoctoral M.D./Ph.D. fellowship (F30 CA203446).
F.N. and N.A. were supported by the Postdoctoral Fellowship for Research Abroad
from the Japan Society for the Promotion of Science (JSPS). A.L is supported by NCI
Individual Postdoctoral Fellowship (F32), Ruth L. Kirschstein National Research Service
Award (NCI 1F32CA20277).
Author contributions
M.M. designed the study, performed most of the experiments, and analyzed data; A.H.Z.
advised on experiment design, developed and performed denervation surgeries, imaged
and quantified prostate innervation, and helped with Alzet pump implantations. H.P.
advised on experiment design, performed blood CFU-C experiments and helped with
bone marrow transplantations and Alzet pump implantations. S.P. advised on experiment
design and helped with HSC transplantations. F.N. helped with sorting and with
CFU-F and mesensphere cultures. N.A. helped with HSC imaging and quantification
of HSC distributions. Q.W. and J.X. analyzed RNA-seq data. X.W. and P.C. helped with
experiments. A.L. helped with image quantification analysis. P.S.F supervised the study.
M.M and P.S.F interpreted data and wrote the manuscript. All authors discussed the
results and commented on the manuscript.
Competing interests
The authors declare no competing financial interests.
Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/
s41591-018-0030-x.
Reprints and permissions information is available at www.nature.com/reprints.
Correspondence and requests for materials should be addressed to P.S.F.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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Articles
Methods
Mice. Nestin-GFP mice24 were bred and aged in our facilities. Aged C57BL/6 mice
were obtained from the National Institute of Aging (NIA), by obtaining retired
breeders from Jackson Laboratory, or by aging young mice (C57BL/6J; Jackson
Laboratory) in our facilities for 8, 12, and 24 months. Adrb3−/− mice were from
the Jackson Laboratory (FVB/N-Adrb3tm1Lowl/J; stock: 006402) and backcrossed to
C57BL/6J background in our facilities. JaB6-Ly5.1 (CD45.1) mice were purchased
from the National Cancer Institute or the Jackson laboratory (B6.SJL-Ptprca Pepcb/
BoyJ). Unless indicated otherwise, 8- to 10-week-old male and female mice were
used as young controls and 20- to 24-month-old males and females were used as
old mice. For all analytical and therapeutic experiments, sex-matched animals from
the same age group were randomly assigned to experimental groups. This study
complied with all ethical regulations involving experiments with mice, and the
Animal Care and Use Committee of Albert Einstein College of Medicine approved
all experimental procedures.
Competitive HSC and bone marrow transplantation. Competitive repopulation
assays were performed using the CD45.1/CD45.2 congenic system. CD45.1
recipient mice were lethally irradiated (12 Gy, two split doses) in a Cesium Mark
1 irradiator (JL Shepherd & Associates). For competitive repopulation assays,
1 ×​  106 CD45.2 donor-nucleated bone marrow cells (BMNCs) were transplanted
into irradiated recipients together with 1 ×​  106 of CD45.1 BMNCs (equal numbers
of cells were pooled from donor mice). For competitive HSC repopulation assays,
200 donor HSCs (CD45.2) were sorted from pooled donor bone marrow cells
and transplanted into irradiated CD45.1 recipients together with a corresponding
number of CD45.1 competitor BMNCs calculated to contain 200 HSCs (1:1 HSC
ratio). For secondary bone marrow transplantation, 3 ×​  106 BMNCs from primary
recipient chimeric mice were transplanted into lethally irradiated CD45.1 young
recipient mice. CD45.1/CD45.2 chimerism of recipient blood and bone marrow
was analyzed up to 5 months after transplantation using FACS analysis.
Reciprocal bone marrow transplantation. For reciprocal bone marrow
transplantation, 4 ×​  106 BMNCs from 2-month-old CD45.1 donor mice were
transplanted into Adrb3+/+ and Adrb3−/− CD45.2 two-month-old recipient mice that
had been lethally irradiated (12 Gy, two split doses) in a Cesium Mark 1 irradiator
(JL Shepherd & Associates). CD45.1/CD45.2 donor chimerism of recipient blood
and bone marrow was analyzed 3 months after transplantation (5 months old) and
mice were analyzed accordingly using FACS analysis.
Sample preparation for flow cytometry and cell sorting. Nucleated single-
cell suspensions enriched from bone marrow were obtained by flushing and
dissociating BM plugs using a 21-gauge needle in phosphate-buffered saline (PBS;
Gibco). Single-cell suspensions enriched from peripheral blood were obtained by
retro-orbital or cheek bleeding of mice. For analysis of stromal and endothelial
cell populations, intact flushed bone marrow plugs were digested for 30 min at
37 °C in 1 mg mL−1 collagenase type IV (Gibco) and 2 mg mL−1 Dispase (Gibco) in
Hank’s balanced salt solution (HBSS; Gibco). Compact bones (after flushing out
marrow) were crushed and digested in 3 mg mL−1 collagenase type 1A (Sigma)
in 1% FBS/HSBSS for 90 min, rotating at 37 °C. For FACS analysis or sorting,
cells were stained with antibodies in PEB (PBS containing 0.5% BSA and 2 mM
EDTA). Dead cells and debris were excluded by FSC (forward scatter), SSC (side
scatter) and DAPI (4′​,6-diamino-2-phenylindole; Sigma) staining profiles. To
eliminate the nonendothelial CD31+ fraction, total ECs quantified by FACS were
identified as CD45−Ter119−CD31highSca-1+, and arteriolar ECs were identified
as CD45−Ter119−CD31highSca-1high. For sorting ECs, anti-CD31-Alexa Fluor-647
(MEC13.3; 102516) was injected intravenously 10 min before the mice were killed
for analysis. FACS analyses were carried out using BD LSRII flow cytometer
(BD Biosciences) and cell-sorting experiments were performed using either a BD
FACSAria II Cell Sorter (BD Biosciences) or a MoFlo Astrios (Beckman Coulter).
Data were analyzed with FlowJo 10.0.8 (LLC) or FACS Diva 6.1 software
(BD Biosciences).
Flow cytometry antibodies. The following antibodies were used for flow
cytometry: anti-Ly6A/E-Alexa Fluor-700 (D7; 56-5981-82), anti-CD117-PE/Cy7
(2B8; 105814), anti-CD45.2-PE (104; 109807), CD45–Pacific Blue (30-F11; 103126),
anti-CD31-Alexa Fluor-647 (MEC13.3; 102516), anti-VE-cadherin (CD144)-
APC-Alexa Fluor-647 (BV13; 138006), anti-CD150-PE (TC15-12F12.2; 115904),
anti-Ter119-Pacific Blue (TER-119; 116232), and anti-CD16/32 (Fcγ​R III/II)-APC/
Cy7 (93; 101328), all purchased from BioLegend (San Diego, CA); and anti-CD48-
PerCp-eFluor710 (HM48-1; 46-0481-182), anti-CD45.1-FITC (A20; 11-0453-85),
anti-CD4-PE/Cy7 (GK1.5; 25-0041-82), anti-CD8a-PE/Cy7 (53-6.7; 25-0081-85),
anti-B220-APC-eFluor 780 (RA3-6B2; 47-0452-82), anti-CD11b (Mac-1)-APC-
eFluor 780 (M1/70; 47-0112-82), anti-Gr-1 (Ly-6G)-APC-eFluor 780 (RB6-8C5;
47-5931-82), anti-CD3e-APC-eFluor 780 (145-2C11; 47-0031-82), anti-CD45-APC-
eFluor 780 (30-F11; 47-0451-82), anti-CD31-PE/Cy7 (MEC13.3; 25-0311-82), anti-
PDGFRα​(CD140a)-APC (APA5; 17-1401-81), anti-PDGFRα​ (CD140a)-PE/Cy7
(APA5; 25-1401-82), CD51-PE (RMV-7; 13-0512-85), anti-CD41-FITC (MWReg30;
11-0411-81), anti-Ki-67-PerCp-eFluor 710 (SolA15; 46-5698-80), anti-Ter119-APC-
eFluor 780 (TER-119; 47-5921-82), streptavidin APC-eFluor 780 (47-4317-82),
© 2018 Nature America Inc., part of Springer Nature. All rights reserved.
Nature Medicine
anti-CD34-eFluor 660 (RAM34; 50-0341-82 at 1:50 dilution), and anti-CD127
(IL7R)-PerCp (A7R34; 45-1271-80), CD135 (Flt3)-PE (A2F10; 12-1351-83), all
purchased from eBioscience (Thermo Fisher). The anti-lineage panel cocktail
(TER-119, RB6-8C5, RA3-6B2, M1/70, 145-2C11, at 1:50 dilution) was from BD
Biosciences (559971). Unless otherwise specified, all antibodies were used at a
1:100 dilution. All antibodies were anti-mouse and their specificity was validated in
previous studies performed in our laboratory24,27,35,42,53,60.
In vitro culture assays. The hematopoietic progenitor colony-forming units in
culture (CFU-C) assay was done as previously described60. HSC culture was done
as previously described27, in the presence or absence of 10, 25, 50, or 100 µ​M
BRL37344 (Sigma). MSC culture, CFU-F, and mesensphere formation assays
were using freshly sorted CD45−Ter119−CD31−CD51+PDGFRα​+ MSCs from
digested bone marrow or CD45−Ter119−CD31−Nestin-GFP+ MSCs from digested
compact bone, plated at clonal densities in 6-well plates (CFU-F: 1,000 cells/well;
mesenspheres: 3,000 cells/well) under culture conditions previously described42.
Cells were cultured for 12–14 d, at the end of which the colonies and spheres were
scored. For CFU-C and mesensphere cultures, low-adhesion tissue culture plates
(Stem Cell Technologies) were used.
Cell cycle analysis. Analyses of the cell cycle were performed as described
previously24. Briefly, single-cell suspension was stained for cell surface markers and
subsequently fixed with Cytofix/Cytoperm fixation and permeabilization solution
(BD Biosciences), according to the manufacturer’s instructions. The cells were then
stained with anti-Ki-67 antibody (eBioscience) and Hoechst 33342 (Sigma) at
20 µ​g mL−1 for 30 min.
Bone marrow denervation by transaction of femoral and sciatic nerves.
Denervation of the femoral and tibial bone marrow was done as previously
described45,46. The femoral nerve was localized after its exit from the vertebral
column deep in the psoas muscle. This was accomplished with a midline
abdominal incision; the intestines were gently moved aside to visualize the psoas
muscle. An incision was made in the psoas to visualize the femoral nerve, and a
1-cm section of the nerve was excised. Deep to the psoas (through the incision),
the sciatic nerve was visualized in close proximity to the iliac crest of the pelvis,
and a 1-cm section of the nerve was excised. For sham operation, both femoral and
sciatic nerves were exposed by surgery, but were left intact.
Musculocutaneous sensory and motor denervation. As previously descried50, to
denervate the hindlimb to induce immobilization without affecting bone marrow
innervation, 1 cm of the sciatic nerve was transacted distal to the spinal cord and
close to the sciatic notch, and 1 cm of the femoral nerve was transacted distal to the
spinal cord after exiting the abdomen inferior to the inguinal ligament. For sham
operation, same sites of both femoral and sciatic nerves were exposed by surgery,
but left intact.
Immunofluorescence staining and imaging of whole-mount sternal
tissues and frozen sections of long bones. Whole-mount tissue preparation,
immunofluorescence staining, and imaging of the sternum were performed as
described previously24,53. Antibodies used for HSC stains are: anti-lineage panel
cocktail (TER-119, RB6-8C5, RA3-6B2, M1/70, 145-2C11 at 1:50 dilution) from
BD Biosciences (559971); anti-CD48-biotin (HM48-1; 13-0481-80, 1:100 dilution),
and CD41-biotin (MWReg30; 11-0411-82, 1:2,500 dilution) from eBioscience
(Thermo Fisher); and anti-CD31-Alexa Fluor 647 (MEC13.3; 102516), anti-CD144
(VE-cadherin)-APC-Alexa Fluor 647 (BV13; 138006), and anti-CD150-PE (TC15-
12F12.2; 115904 at 1:100 dilution) from Biolegend (San Diego, CA). For all imaging
experiments, anti-CD31 and anti-CD144 antibodies were injected intravenously
to mice (10 μ​g, 20 μ​L of 0.5 μ​g μ​L−1) and mice were killed for analysis 10 min after
injection. For frozen sections of long bones, femurs and tibias were fixed in 4%
paraformaldehyde (PFA) overnight at 4 °C. For cryopreservation, the bones were
incubated sequentially in 10%, 20%, and 30% sucrose/PBS at 4 °C for 1 h each,
and embedded and flash frozen in SCEM embedding medium (SECTION-LAB).
Frozen sections were prepared 20 µ​m thick with a Cryostat (CM3050, Leica) using
Kawamoto’s tape transfer method61. For immunofluorescence staining, sections
were rinsed with PBS, postfixed with 4% cold PFA for 10 min followed by blocking
with 20% donkey serum (DS; Sigma) in 0.5% Triton X-100/PBS for 3 h at room
temperature (20–25 °C). For SNS nerve staining, anti-tyrosine hydroxylase (TH)
antibody (cat: AB152; lot: 2493925; Millipore) was used at 1:100 dilution in 2%
DS 0.1% Triton X-100/PBS for 72 h at 4 °C. For pan-neural staining, Alexa Fluor
488-conjugated anti-tubulin β​3 (Tubb3) antibody (clone: AA10; cat: 657404;
Biolegend) was used at 1:100 dilution in 2% DS 0.1% Triton X-100/PBS for 72 h
at 4 °C. For synaptophysin staining, rabbit polyclonal anti-synaptophysin (cat:
ab32594; Abcam) antibody was used at 1:100 dilution in 2% DS 0.1% Triton X-100/
PBS for 72 h at 4 °C. For perilipin staining, rabbit polyclonal anti-perilipin antibody
(clone: D1D8; Cat: 9349; Cell Signaling Technology) was used at 1:100 dilution in
2% DS 0.1% Triton X-100/PBS overnight at 4 °C. For α​-SMA staining, monoclonal
anti-actin α​-smooth muscle-Cy3-conjugated antibody (clone: 1A4; cat: C6198;
Sigma) was used at 1:100 dilution in 2% DS 0.1% Triton X-100/PBS for 30 min at
room temperature (20–25 °C). When necessary, primary antibody staining was
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Nature Medicine
followed by 3 washes with 2% DS 0.1% Triton X-100/PBS and 30 min incubation
with Alexa Fluor 568-conjugated goat anti-rabbit IgG (cat: A11011, Invitrogen)
secondary antibody and 0.2% DAPI (4′​,6-diamino-2-phenylindole; Sigma).
All images were acquired at room temperature using a Zeiss Axio examiner D1
microscope (Zeiss) with confocal scanner unit (Yokogawa), and reconstructed
in three dimensions with Slide Book software (Intelligent Imaging Innovations).
Image analysis was performed using both Slide Book software (Intelligent Imaging
Innovations) and the Fiji build of ImageJ (NIH).
Immunofluorescence staining and imaging of sorted HSCs. Immunofluorescence
staining for either γ​H2AX or Cdc42 and tubulin was adapted from10,16.
Lineage−CD48−Sca-1+c-Kit+CD150+ HSCs were directly sorted on polylysine
coated slides (P0425-72AE; Sigma), 500–2,000 cells per slide, incubated for 10 min,
fixed with 4% PFA for 10 min at room temperature (20–25 °C), and permeabilized
with 0.2% Triton X-100 for 20 min at room temperature. Consequently, the cells
were blocked with 1% BSA/PBS overnight at 4 °C. For γ​H2AX staining, cells were
incubated with 0.125 μ​g (5 μ​L of 25 μ​g mL−1) FITC-conjugated anti-phospho-H2A.X
(ser139) antibody (clone: 2F3; cat: 613404; Biolegend) for 2 h at 37 °C. Primary
antibody staining was followed by 3 washes with PBS and 10 min incubation with
0.2% DAPI (Sigma). For Cdc42 and tubulin staining, cells were incubated with
rabbit polyclonal anti-Cdc42 antibody (1:50 dilution, cat: 07-1466; Millipore) and
rat monoclonal anti-tubulin antibody (1:100 dilution, clone: YL1/2; cat: ab6160;
Abcam) for 2 h at 37 °C. Primary antibody staining was followed by 3 washes with
PBS and 1 h incubation at room temperature with Alexa Fluor 633-conjugated goat
anti-rabbit IgG (cat: A21071; Invitrogen) and Alexa Fluor 488-conjugated goat anti-
rat IgG (cat: A11006; Invitrogen) both at 1:100 dilution. Secondary antibody staining
was followed by 3 washes with PBS and 10 min incubation with 0.2% DAPI (Sigma).
All images were acquired using a Zeiss Axio examiner D1 microscope (Zeiss) with
confocal scanner unit (Yokogawa), and reconstructed in three dimensions with Slide
Book software (Intelligent Imaging Innovations). Image analysis was performed
using Slide Book (Intelligent Imaging Innovations).
RNA isolation and quantitative real time PCR (q-PCR). RNA isolation and
q-PCR were performed as described previously24. All mRNA expression levels were
calculated relative to Gapdh or Actb. To ascertain that the expression levels of the
housekeeping genes did not change with age, for all experiments, Actb mRNA levels
were quantified relative to Gapdh and did not show any changes. The sequences of
the oligonucleotides used can be provided upon reasonable request.
RNA-seq and analysis. Total RNA from 2,000 sorted HSCs was extracted using
the RNeasy Plus Micro kit (Qiagen), and assessed for integrity and purity using an
Aligent 2100 Bioanalyzer (Agilent Technologies). Complementary DNA libraries
were then generated using the SMART-Seq v4 Ultra Low Input RNA Kit for
Sequencing (Clontech Laboratories) and the Nextera XT DNA Sample Preparation
Kit (Illumina). The libraries were then submitted for Illumina NextSeq 500
sequencing (150-bp, single-ended) according to standard procedures (ENSEMBL
Mus musculus reference genome GRCm38, release 77). RNA-seq data generated
from Illumina NextSeq 500 were processed using the following pipeline. Briefly,
single- and double-ended sequencing reads were aligned to the mouse genome
(mm10) using Spliced Transcripts Alignment to a Reference (STAR 2.5.3a).
Aligned reads were then quantified to transcriptome (mm10; Ensembl Transcripts
release 83) and normalized. Differentially expressed genes were identified using
Partek Flow. Hierarchical clustering, and Gene Set Enrichment Analysis (GSEA)62,63
was then performed on the top 150 variable genes.
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© 2018 Nature America Inc., part of Springer Nature. All rights reserved.
Articles
In vivo administration of β-adrenergic agonists. For long-term administration
of β​-adrenergic agonists, clenbuterol (Sigma) and BRL37344 (Sigma) were dosed
at 2 mg kg−1 d−1 and 2.4 mg kg−1 d−1, respectively, using subcutaneously implanted
Alzet osmotic pumps (model 2006; DURECT Corporation). Six weeks following
implantation of the first pump, an additional pump was implanted for a total of
12 weeks of continuous drug delivery. Control animals were implanted with Alzet
pumps containing saline (PBS; Gibco).
Statistics and reproducibility. All data are represented as mean ±​  s.e.m. No
statistical method was used to predetermine sample sizes; sample sizes were
determined by previous experience with similar models of hematopoiesis,
as shown in previous experiments done in our laboratory23,24,27,35,42,53. For all
experiments, mice were randomly assigned to experimental groups and no sample
blinding was used for experiment collection or analysis. Sample exclusion was only
done as a result of premature mouse death or infection due injury or improper
healing postsurgery. Experiments presented were successfully reproduced at least
three times. For box plots, box span from the 25th to 75th percentiles and the
centerline is plotted at the median. Whiskers represent minimum to maximum
range. Comparisons between two groups were done using unpaired two-tailed
t tests and paired two-tailed t tests for denervation experiments. The t ratio was
calculated as difference between sample means divided by the standard error of
the difference and calculated by combining the s.e.m. of the two groups. Degrees
of freedom (df) were calculated as total sample size – 2 (t values and degrees of
freedom are provided in source data). One-way ANOVA with Tukey’s multiple
comparisons tests were used for multiple group comparisons; two-way ANOVA
with Bonferroni’s multiple comparison tests were used for multiple-parameter
analysis, and two-sample Kolmogorov–Smirnov tests were used for comparisons
of distribution patterns. Data statistical analyses and presentation were performed
using GraphPad Prism 7 (GraphPad Software, San Diego, CA), FACS Diva 6.1
software (BD Biosciences), FlowJo 10.0.8 (LLC), Slide Book (Intelligent Imaging
Innovations), and Fiji build of ImageJ (NIH).
Reporting summary. Further information on experimental design is available in
the Nature Research Reporting Summary.
Data availability. RNA sequencing data have been deposited in the Gene
Expression Omnibus under accession number GSE109546. Source data are
available for Figs. 1–5. All other source data are available upon reasonable request
from the corresponding author.
References
60. Pierce, H. et al. Cholinergic signals from the CNS regulate G-CSF-mediated
HSC mobilization from bone marrow via a glucocorticoid signaling relay.
Cell Stem Cell 20, 648–658.e4 (2017).
61. Kawamoto, T. & Shimizu, M. A method for preparing 2- to 50-micron-thick
fresh-frozen sections of large samples and undecalcified hard tissues.
Histochem. Cell Biol. 113, 331–339 (2000).
62. Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based
approach for interpreting genome-wide expression profiles. Proc. Natl. Acad.
Sci. USA 102, 15545–15550 (2005).
63. Mootha, V. K. et al. PGC-1α​-responsive genes involved in oxidative
phosphorylation are coordinately downregulated in human diabetes.
Nat. Genet. 34, 267–273 (2003).
 nature research | life sciences reporting summary
Corresponding author(s): Paul S. Frenette
Initial submission Revised version Final submission

Life Sciences Reporting Summary

Nature Research wishes to improve the reproducibility of the work that we publish. This form is intended for publication with all accepted life
science papers and provides structure for consistency and transparency in reporting. Every life science submission will use this form; some list
items might not apply to an individual manuscript, but all fields must be completed for clarity.
For further information on the points included in this form, see Reporting Life Sciences Research. For further information on Nature Research
policies, including our data availability policy, see Authors & Referees and the Editorial Policy Checklist.

` Experimental design
1. Sample size
Describe how sample size was determined. No statistical method was used to predetermine sample size. Sample size was
predetermined according to statistical data analysis of previous studies utilizing
similar models of hematopoiesis done in our laboratory. See citations in the
Statistics parts of the Methods section in the manuscript.
2. Data exclusions
Describe any data exclusions. Sample exclusion was done only as a result of premature mouse death or infection
due injury or improper healing post surgery.
3. Replication
Describe whether the experimental findings were All attempts of replication were successful.
reliably reproduced.
4. Randomization
Describe how samples/organisms/participants were Mice were randomly assigned to experimental groups. All experiments were
allocated into experimental groups. repeated in both male and female mice. For all experiments comparing mice at
different ages, age and sex matched mice were randomly assigned to each test
group.
5. Blinding
Describe whether the investigators were blinded to No sample blinding was utilized for either data collection or analysis. Data blinding
group allocation during data collection and/or analysis. was not possible for most experiments due to obvious difference in mouse size and
weight between young and old mice. Denervation experiments compared samples
derived from the same mouse (denervated versus sham limb)
Note: all studies involving animals and/or human research participants must disclose whether blinding and randomization were used.

June 2017

1
6. Statistical parameters

nature research | life sciences reporting summary

For all figures and tables that use statistical methods, confirm that the following items are present in relevant figure legends (or in the
Methods section if additional space is needed).

n/a Confirmed

The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement (animals, litters, cultures, etc.)
A description of how samples were collected, noting whether measurements were taken from distinct samples or whether the same
sample was measured repeatedly
A statement indicating how many times each experiment was replicated
The statistical test(s) used and whether they are one- or two-sided (note: only common tests should be described solely by name; more
complex techniques should be described in the Methods section)
A description of any assumptions or corrections, such as an adjustment for multiple comparisons
The test results (e.g. P values) given as exact values whenever possible and with confidence intervals noted
A clear description of statistics including central tendency (e.g. median, mean) and variation (e.g. standard deviation, interquartile range)
Clearly defined error bars

See the web collection on statistics for biologists for further resources and guidance.

` Software
Policy information about availability of computer code
7. Software
Describe the software used to analyze the data in this Data analysis and presentation was performed using either GraphPad Prism 7
study. (GraphPad Software, San Diego, CA), FACS Diva 6.1 software (BD Biosciences),
FlowJo 10.0.8 (LLC), Slide Book software (Intelligent Imaging Innovations) and Fiji
build of ImageJ (NIH). All statistical analyses were performed using GraphPad Prism
7.
For manuscripts utilizing custom algorithms or software that are central to the paper but not yet described in the published literature, software must be made
available to editors and reviewers upon request. We strongly encourage code deposition in a community repository (e.g. GitHub). Nature Methods guidance for
providing algorithms and software for publication provides further information on this topic.

` Materials and reagents

Policy information about availability of materials
8. Materials availability
Indicate whether there are restrictions on availability of All unique materials used in this study are available upon request. No restrictions
unique materials or if these materials are only available apply.
for distribution by a for-profit company.

June 2017

2
9. Antibodies

nature research | life sciences reporting summary

Describe the antibodies used and how they were validated The following antibodies were used for flow cytometry: anti-Ly6A/E-Alexa Fluor
for use in the system under study (i.e. assay and species). 700 (D7; 56-5981-82), anti-CD117-PE/Cy7 (2B8; 105814), anti-CD45.2-PE (104;
109807), CD45- Pacific Blue (30-F11; 103126), anti-CD31-Alexa Fluor 647
(MEC13.3; 102516), anti-VE-cadherin (CD144)-APC-Alexa Fluor 647 (BV13;
138006), Anti-CD150-PE (TC15-12F12.2; 115904), anti-Ter119-Pacific Blue
(TER-119; 116232), anti-CD16/32 (FcγR III/II)-APC/Cy7 (93; 101328) were
purchased from BioLegend (San Diego, CA). Anti-CD48-PerCp-eFluor710 (HM48-1;
46-0481-182), anti-CD45.1-FITC (A20; 11-0453-85), anti-CD4-PE/Cy7 (GK1.5;
25-0041-82), anti-CD8a-PE/Cy7 (53-6.7; 25-0081-85), anti-B220-APC-eFluor 780
(RA3-6B2; 47-0452-82), anti-CD11b (Mac-1)-APC-eFluor 780 (M1/70; 47-0112-82),
anti-Gr-1 (Ly-6G)- APC-eFluor 780 (RB6-8C5; 47-5931-82), anti-CD3e-APC-eFluor
780 (145-2C11; 47-0031-82), anti-CD45-APC-eFluor 780 (30-F11; 47-0451-82), anti-
CD31-PE/Cy7 (MEC13.3; 25-0311-82), anti-PDGFRα (CD140a)-APC (APA5;
17-1401-81), anti-PDGFRα (CD140a)-PE/Cy7 (APA5; 25-1401-82), CD51-PE (RMV-7;
13-0512-85), anti-CD41-FITC (MWReg30; 11-0411-81), anti-Ki-67-PerCp-eFluor 710
(SolA15; 46-5698-80), anti-Ter119-APC-eFluor 780 (TER-119; 47-5921-82),
Streptavidin APC-eFluor 780 (47-4317-82), anti-CD34-eFluor 660 (RAM34;
50-0341-82 at 1:50 dilution), anti-CD127 (IL7R)-PerCp (A7R34; 45-1271-80), CD135
(Flt3)-PE (A2F10; 12-1351-83) were purchased from eBioscience (Thermo Fisher).
Anti-lineage panel cocktail (TER-119, RB6-8C5, RA3-6B2, M1/70, 145-2C11 at 1:50
dilution) was from BD Biosciences (559971). Unless otherwise specified, all
antibodies were used at a 1:100 dilution.
Antibodies used for immunofluorescence imaging: Anti-lineage panel cocktail
(TER-119, RB6-8C5, RA3-6B2, M1/70, 145-2C11 at 1:50 dilution) from BD
Biosciences (559971), anti-CD48-biotin (HM48-1; 13-0481-80, 1:100 dilution),
CD41-biotin (MWReg30; 11-0411-82, 1:2500 dilution) from eBioscience (Thermo
Fisher) and anti-CD31-Alexa Fluor 647 (MEC13.3; 102516), anti-CD144 (VE-
cadherin)-APC-Alexa Fluor 647 (BV13; 138006), Anti-CD150-PE (TC15-12F12.2;
115904 at 1:100 dilution) from Biolegend (San Diego, CA). Anti-Tyrosine
Hydroxylase (TH) antibody (Cat: AB152; Lot: 2493925; Millipore), Alexa Fluor 488
conjugated anti-Tubulin Beta3 (Tubb3) antibody (Clone: AA10; Cat: 657404;
Biolegend), rabbit polyclonal anti-Synaptophysin (Cat: ab32594; Abcam), rabbit
polyclonal anti-Perilipin antibody (Clone: D1D8; Cat: 9349; Cell Signaling
Technology), monoclonal anti-Actin α-Smooth Muscle - Cy3-conjugated antibody
(Clone: 1A4; Cat: C6198; Sigma), Alexa Fluor 568-conjugated goat anti-rabbit IgG
(Cat: A11011, Invitrogen), FITC-conjugated anti-phospho H2A.X (ser139) antibody
(Clone: 2F3; Cat: 613404; Biolegend), rabbit polyclonal anti-Cdc42 antibody (1:50
dilution, Cat: 07-1466; Millipore), rat monoclonal anti-Tubulin antibody (1:100
dilution, Clone: YL1/2; Cat: ab6160; Abcam), Alexa Fluor 633-conjugated goat anti-
rabbit IgG (Cat: A21071; Invitrogen) and Alexa Fluor 488-conjugated goat anti-rat
IgG (Cat: A11006; Invitrogen).

All flow cytometry antibodies were anti-mouse and their specificity was validated
in previous studies performed in our laboratory.
Most imaging antibodies were validated in previous studies done by our
laboratory, the specificity of other antibodies was validated by comparing antibody
staining with staining of isotope control antibodies and secondary antibody only
staining.
Please citations in the Online Methods part of the manuscript for our previous
studies using similar antibodies.

10. Eukaryotic cell lines

a. State the source of each eukaryotic cell line used. Eukaryotic cell lines were not used in the study.

b. Describe the method of cell line authentication used. N/A

c. Report whether the cell lines were tested for N/A

mycoplasma contamination.
June 2017

d. If any of the cell lines used are listed in the database N/A
of commonly misidentified cell lines maintained by
ICLAC, provide a scientific rationale for their use.

3
` Animals and human research participants
Policy information about studies involving animals; when reporting animal research, follow the ARRIVE guidelines
11. Description of research animals
Provide details on animals and/or animal-derived
materials used in the study.
Policy information about studies involving human research participants
12. Description of human research participants
Describe the covariate-relevant population
characteristics of the human research participants.
nature research | life sciences reporting summary
Nestin–GFP mice were bred and aged in our facilities. Aged C57BL/6 mice were
obtained from the National Institute of Aging (NIA), or retired breeders from
Jackson Laboratory, or by aging mice in our facilities for 8, 12 and 24 months.
Young C57BL/6 mice were purchased from the National Cancer Institute or the
Jackson laboratory (C57Bl/6J). Adrb3−/− mice were from the Jackson Laboratory
(FVB/N-Adrb3tm1Lowl/J; stock: 006402) and back-crossed to C57BL/6 background
in our facilities. JaB6-Ly5.1 (CD45.1) mice were purchased from the National
Cancer Institute or the Jackson laboratory (B6.SJL-Ptprca Pepcb/BoyJ). Unless
indicated otherwise, 8 to 10-week-old male and female mice were used as young
controls and 20 to 24-month-old males and females were designated as old mice.
For all analytical and therapeutic experiments, sex-matched animals from the same
age group were randomly assigned to experimental groups.
This study did not involve human research participants
June 2017
4
 nature research | flow cytometry reporting summary
Corresponding author(s): Paul S. Frenette
Initial submission Revised version Final submission

Flow Cytometry Reporting Summary

Form fields will expand as needed. Please do not leave fields blank.

` Data presentation
For all flow cytometry data, confirm that:
1. The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
2. The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of
identical markers).
3. All plots are contour plots with outliers or pseudocolor plots.
4. A numerical value for number of cells or percentage (with statistics) is provided.

` Methodological details
5. Describe the sample preparation.
Nucleated single-cell suspensions enriched from bone marrow were
obtained by flushing and dissociating BM plugs using a 21G needle in
phosphate-buffered saline (PBS; Gibco). Single-cell suspensions enriched
from peripheral blood were obtained by retro orbital or cheek bleeding of
mice. For analysis of stromal and endothelial cell populations, intact
flushed bone marrow plugs were digested for 30 min at 37°C in 1 mg ml-1
Collagenase type IV (Gibco) and 2 mg ml-1 Dispase (Gibco) in Hank’s
balanced salt solution (HBSS; Gibco). Compact bones (after flushing out
marrow) were crushed and digested in 3 mg ml-1 Collagenase type 1A
(Sigma) in 1% FBS/HSBSS for 90 min rotating at 37°C. For FACS analysis or
sorting, cells were stained with antibodies in PEB (PBS containing 0.5% BSA
and 2 mM EDTA). Dead cells and debris were excluded by FSC, SSC and
DAPI (4', 6-diamino-2-phenylindole; Sigma) staining profiles. To eliminate
the non-endothelial CD31+ fraction, total ECs quantified by FACS were
identified as CD45− Ter119− CD31high Sca-1+ and arteriolar ECs were
identified as CD45− Ter119− CD31high Sca-1high. For sorting ECs, anti-
CD31-Alexa Fluor 647 (MEC13.3; 102516) was injected intravenously 10
min prior to sacrificing mice.

6. Identify the instrument used for data collection.
BD LSRII Special Order System (BD Bioscience) was used for all data
acquisition (H55100027).
BD FACSAria IIu (P46900051) and BD FACSAria II (P69500137) Special
Order Systems were used for most sorting experiments.
MoFlo Astrios EQ (Beckman coulter) was used for sorting HSCs for RNA-seq
analysis and imaging.

7. Describe the software used to collect and analyze Data was collected using BD FACSDiva 6.1 (BD Biosciences) software
the flow cytometry data. Data was analyzed with FACSDiva 6.1 and FlowJo V.10.1 (LLC) softwares

8. Describe the abundance of the relevant cell Purity of cells sorted or analyzed was determined by their appropriate
populations within post-sort fractions. frequency and absolute numbers determined for control wild-type young
June 2017

mice according to previous studies. It was described in the literature that

HSCs comprise around 0.01% of total bone marrow. We have previously
shown that MSCs and ECs are 0.05% and 0.05-0.1% respectively, of total
bone marrow. In every experiment, we included a control group
comprised of a cohort of young control mice.
Because the populations we were sorting are very rare, we did not do post

1
 sort analysis.

9. Describe the gating strategy used. For all flow cytometric analysis and sorting debris were excluded by FSC,

nature research | flow cytometry reporting summary

SSC scatters and DAPI (4', 6-diamino-2-phenylindole) staining was used to
exclude dead cells, following which specific population were gated
according to prior experience with doing similar experiments in our
laboratory . Briefly, for mesenchymal stem cell (MSC) gating: CD45+
Ter119+ CD31+ cells were excluded by staining with CD45, Ter119 APC-
Cy7 or Pacific Blue and CD31-PE-Cy7. MSCs were identified, as previously
described, to be CD51-PE+ and CD140a (PDGFRa)-APC positive within the
CD45- Ter119- CD31- triple negative population. Gates were determined
by using specific fluorescence minus one (FMO) controls.
Endothelial cells (ECs) were identified as CD45- Ter119- and positive for
CD31-PE-Cy7 and Sca-1 - Alexa Fluor 700. To eliminate the non-endothelial
CD31+ fraction, total ECs quantified by FACS were identified as CD45−
Ter119− CD31high Sca-1+ and arteriolar ECs were identified as CD45−
Ter119− CD31high Sca-1high.
Detailed gating strategies for stromal cell populations and cell cycle
analysis are presented in Fig. 1f, Fig. 3d, Fig. 3e, Fig. 4h, Supplementary
Fig. 1a, Supplementary Fig. 7a and Supplementary Fig.8f.
Hematopoietic stem cells (HSCs) were identified as previously described in
the literature: negative for lineage (CD3e, B220, Gr-1, CD11b, Ter119)-
APC-Cy7, negative for CD48-PerCp and positive for Sca-1-Alexa fluor 700,
CD117 (c-Kit)-PE-Cy7 and CD150-PE (SLAM markers)
All other FACS analyses - Ki67 positivity, donor derived CD45.2 cells in
transplantation setting, myeloid cells (CD11b+), B cells (B220+), T cells
(CD4+/CD8+) have all been described previously (see manuscript for
citations describing similar studies done in our laboratory)
Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.

June 2017