• Aging is associated with morphologic changes in HSPCs and megakaryocytes.

  • HSPCs lie proximal to the bone, vasculature, and megakaryocytes in young BM but further from megakaryocytes in old BM.

Abstract

The spatial anatomy of hematopoiesis in the bone marrow (BM) has been extensively studied in mice and other preclinical models, but technical challenges have precluded a commensurate exploration in humans. Institutional pathology archives contain thousands of paraffinized BM core biopsy tissue specimens, providing a rich resource for studying the intact human BM topography in a variety of physiologic states. Thus, we developed an end-to-end pipeline involving multiparameter whole tissue staining, in situ imaging at single-cell resolution, and artificial intelligence–based digital whole slide image analysis and then applied it to a cohort of disease-free samples to survey alterations in the hematopoietic topography associated with aging. Our data indicate heterogeneity in marrow adipose tissue (MAT) content within each age group and an inverse correlation between MAT content and proportions of early myeloid and erythroid precursors, irrespective of age. We identify consistent endosteal and perivascular positioning of hematopoietic stem and progenitor cells (HSPCs) with medullary localization of more differentiated elements and, importantly, uncover new evidence of aging-associated changes in cellular and vascular morphologies, microarchitectural alterations suggestive of foci with increased lymphocytes, and diminution of a potentially active megakaryocytic niche. Overall, our findings suggest that there is topographic remodeling of human hematopoiesis associated with aging. More generally, we demonstrate the potential to deeply unravel the spatial biology of normal and pathologic human BM states using intact archival tissue specimens.

1.
Jagannathan-Bogdan
M
,
Zon
LI
.
Hematopoiesis
.
Dev Camb Engl
.
2013
;
140
(
12
):
2463
-
2467
.
2.
Pinho
S
,
Frenette
PS
.
Haematopoietic stem cell activity and interactions with the niche
.
Nat Rev Mol Cell Biol
.
2019
;
20
(
5
):
303
-
320
.
3.
Crane
GM
,
Jeffery
E
,
Morrison
SJ
.
Adult haematopoietic stem cell niches
.
Nat Rev Immunol
.
2017
;
17
(
9
):
573
-
590
.
4.
Lo Celso
C
,
Fleming
HE
,
Wu
JW
, et al
.
Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche
.
Nature
.
2009
;
457
(
7225
):
92
-
96
.
5.
Xie
Y
,
Yin
T
,
Wiegraebe
W
, et al
.
Detection of functional haematopoietic stem cell niche using real-time imaging
.
Nature
.
2009
;
457
(
7225
):
97
-
101
.
6.
Méndez-Ferrer
S
,
Michurina
TV
,
Ferraro
F
, et al
.
Mesenchymal and haematopoietic stem cells form a unique bone marrow niche
.
Nature
.
2010
;
466
(
7308
):
829
-
834
.
7.
Kunisaki
Y
,
Bruns
I
,
Scheiermann
C
, et al
.
Arteriolar niches maintain haematopoietic stem cell quiescence
.
Nature
.
2013
;
502
(
7473
):
637
-
643
.
8.
Nombela-Arrieta
C
,
Pivarnik
G
,
Winkel
B
, et al
.
Quantitative imaging of haematopoietic stem and progenitor cell localization and hypoxic status in the bone marrow microenvironment
.
Nat Cell Biol
.
2013
;
15
(
5
):
533
-
543
.
9.
Spencer
JA
,
Ferraro
F
,
Roussakis
E
, et al
.
Direct measurement of local oxygen concentration in the bone marrow of live animals
.
Nature
.
2014
;
508
(
7495
):
269
-
273
.
10.
Acar
M
,
Kocherlakota
KS
,
Murphy
MM
, et al
.
Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal
.
Nature
.
2015
;
526
(
7571
):
126
-
130
.
11.
Hawkins
ED
,
Duarte
D
,
Akinduro
O
, et al
.
T-cell acute leukaemia exhibits dynamic interactions with bone marrow microenvironments
.
Nature
.
2016
;
538
(
7626
):
518
-
522
.
12.
Itkin
T
,
Gur-Cohen
S
,
Spencer
JA
, et al
.
Distinct bone marrow blood vessels differentially regulate haematopoiesis
.
Nature
.
2016
;
532
(
7599
):
323
-
328
.
13.
Duarte
D
,
Hawkins
ED
,
Akinduro
O
, et al
.
Inhibition of endosteal vascular niche remodeling rescues hematopoietic stem cell loss in AML
.
Cell Stem Cell
.
2018
;
22
(
1
):
64
-
77.e6
.
14.
Pinho
S
,
Marchand
T
,
Yang
E
,
Wei
Q
,
Nerlov
C
,
Frenette
PS
.
Lineage-biased hematopoietic stem cells are regulated by distinct niches
.
Dev Cell
.
2018
;
44
(
5
):
634
-
641.e4
.
15.
Saçma
M
,
Pospiech
J
,
Bogeska
R
, et al
.
Haematopoietic stem cells in perisinusoidal niches are protected from ageing
.
Nat Cell Biol
.
2019
;
21
(
11
):
1309
-
1320
.
16.
Kokkaliaris
KD
,
Kunz
L
,
Cabezas-Wallscheid
N
, et al
.
Adult blood stem cell localization reflects the abundance of reported bone marrow niche cell types and their combinations
.
Blood
.
2020
;
136
(
20
):
2296
-
2307
.
17.
Nilsson
SK
,
Dooner
MS
,
Tiarks
CY
,
Weier
HU
,
Quesenberry
PJ
.
Potential and distribution of transplanted hematopoietic stem cells in a nonablated mouse model
.
Blood
.
1997
;
89
(
11
):
4013
-
4020
.
18.
Calvi
LM
,
Adams
GB
,
Weibrecht
KW
, et al
.
Osteoblastic cells regulate the haematopoietic stem cell niche
.
Nature
.
2003
;
425
(
6960
):
841
-
846
.
19.
Zhang
J
,
Niu
C
,
Ye
L
, et al
.
Identification of the haematopoietic stem cell niche and control of the niche size
.
Nature
.
2003
;
425
(
6960
):
836
-
841
.
20.
Nilsson
SK
,
Johnston
HM
,
Whitty
GA
, et al
.
Osteopontin, a key component of the hematopoietic stem cell niche and regulator of primitive hematopoietic progenitor cells
.
Blood
.
2005
;
106
(
4
):
1232
-
1239
.
21.
Adams
GB
,
Chabner
KT
,
Alley
IR
, et al
.
Stem cell engraftment at the endosteal niche is specified by the calcium-sensing receptor
.
Nature
.
2006
;
439
(
7076
):
599
-
603
.
22.
Zhu
J
,
Garrett
R
,
Jung
Y
, et al
.
Osteoblasts support B-lymphocyte commitment and differentiation from hematopoietic stem cells
.
Blood
.
2007
;
109
(
9
):
3706
-
3712
.
23.
Zhao
M
,
Tao
F
,
Venkatraman
A
, et al
.
N-Cadherin-expressing bone and marrow stromal progenitor cells maintain reserve hematopoietic stem cells
.
Cell Rep
.
2019
;
26
(
3
):
652
-
669.e6
.
24.
Kiel
MJ
,
Yilmaz
OH
,
Iwashita
T
,
Yilmaz
OH
,
Terhorst
C
,
Morrison
SJ
.
SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells
.
Cell
.
2005
;
121
(
7
):
1109
-
1121
.
25.
Sugiyama
T
,
Kohara
H
,
Noda
M
,
Nagasawa
T
.
Maintenance of the hematopoietic stem cell pool by CXCL12-CXCR4 chemokine signaling in bone marrow stromal cell niches
.
Immunity
.
2006
;
25
(
6
):
977
-
988
.
26.
Sacchetti
B
,
Funari
A
,
Michienzi
S
, et al
.
Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment
.
Cell
.
2007
;
131
(
2
):
324
-
336
.
27.
Ding
L
,
Saunders
TL
,
Enikolopov
G
,
Morrison
SJ
.
Endothelial and perivascular cells maintain haematopoietic stem cells
.
Nature
.
2012
;
481
(
7382
):
457
-
462
.
28.
Ding
L
,
Morrison
SJ
.
Haematopoietic stem cells and early lymphoid progenitors occupy distinct bone marrow niches
.
Nature
.
2013
;
495
(
7440
):
231
-
235
.
29.
Greenbaum
A
,
Hsu
Y-MS
,
Day
RB
, et al
.
CXCL12 in early mesenchymal progenitors is required for haematopoietic stem-cell maintenance
.
Nature
.
2013
;
495
(
7440
):
227
-
230
.
30.
Chen
JY
,
Miyanishi
M
,
Wang
SK
, et al
.
Hoxb5 marks long-term haematopoietic stem cells and reveals a homogenous perivascular niche
.
Nature
.
2016
;
530
(
7589
):
223
-
227
.
31.
Kusumbe
AP
,
Ramasamy
SK
,
Itkin
T
, et al
.
Age-dependent modulation of vascular niches for haematopoietic stem cells
.
Nature
.
2016
;
532
(
7599
):
380
-
384
.
32.
Asada
N
,
Kunisaki
Y
,
Pierce
H
, et al
.
Differential cytokine contributions of perivascular haematopoietic stem cell niches
.
Nat Cell Biol
.
2017
;
19
(
3
):
214
-
223
.
33.
Comazzetto
S
,
Murphy
MM
,
Berto
S
,
Jeffery
E
,
Zhao
Z
,
Morrison
SJ
.
Restricted hematopoietic progenitors and erythropoiesis require SCF from leptin receptor+ niche cells in the bone marrow
.
Cell Stem Cell
.
2019
;
24
(
3
):
477
-
486.e6
.
34.
Butler
JM
,
Nolan
DJ
,
Vertes
EL
, et al
.
Endothelial cells are essential for the self-renewal and repopulation of Notch-dependent hematopoietic stem cells
.
Cell Stem Cell
.
2010
;
6
(
3
):
251
-
264
.
35.
Yamazaki
S
,
Iwama
A
,
Takayanagi
S
,
Eto
K
,
Ema
H
,
Nakauchi
H
.
TGF-beta as a candidate bone marrow niche signal to induce hematopoietic stem cell hibernation
.
Blood
.
2009
;
113
(
6
):
1250
-
1256
.
36.
Bruns
I
,
Lucas
D
,
Pinho
S
, et al
.
Megakaryocytes regulate hematopoietic stem cell quiescence through CXCL4 secretion
.
Nat Med
.
2014
;
20
(
11
):
1315
-
1320
.
37.
Zhao
M
,
Perry
JM
,
Marshall
H
, et al
.
Megakaryocytes maintain homeostatic quiescence and promote post-injury regeneration of hematopoietic stem cells
.
Nat Med
.
2014
;
20
(
11
):
1321
-
1326
.
38.
Heazlewood
SY
,
Neaves
RJ
,
Williams
B
,
Haylock
DN
,
Adams
TE
,
Nilsson
SK
.
Megakaryocytes co-localise with hemopoietic stem cells and release cytokines that up-regulate stem cell proliferation
.
Stem Cell Res
.
2013
;
11
(
2
):
782
-
792
.
39.
Ludin
A
,
Itkin
T
,
Gur-Cohen
S
, et al
.
Monocytes-macrophages that express α-smooth muscle actin preserve primitive hematopoietic cells in the bone marrow
.
Nat Immunol
.
2012
;
13
(
11
):
1072
-
1082
.
40.
Chow
A
,
Lucas
D
,
Hidalgo
A
, et al
.
Bone marrow CD169+ macrophages promote the retention of hematopoietic stem and progenitor cells in the mesenchymal stem cell niche
.
J Exp Med
.
2011
;
208
(
2
):
261
-
271
.
41.
Winkler
IG
,
Sims
NA
,
Pettit
AR
, et al
.
Bone marrow macrophages maintain hematopoietic stem cell (HSC) niches and their depletion mobilizes HSCs
.
Blood
.
2010
;
116
(
23
):
4815
-
4828
.
42.
Zhou
BO
,
Yu
H
,
Yue
R
, et al
.
Bone marrow adipocytes promote the regeneration of stem cells and haematopoiesis by secreting SCF
.
Nat Cell Biol
.
2017
;
19
(
8
):
891
-
903
.
43.
Sykes
SM
,
Scadden
DT
.
Modeling human hematopoietic stem cell biology in the mouse
.
Semin Hematol
.
2013
;
50
(
2
):
92
-
100
.
44.
Leenaars
CHC
,
Kouwenaar
C
,
Stafleu
FR
, et al
.
Animal to human translation: a systematic scoping review of reported concordance rates
.
J Transl Med
.
2019
;
17
(
1
):
223
.
45.
Watchman
CJ
,
Bourke
VA
,
Lyon
JR
, et al
.
Spatial distribution of blood vessels and CD34+ hematopoietic stem and progenitor cells within the marrow cavities of human cancellous bone
.
J Nucl Med
.
2007
;
48
(
4
):
645
-
654
.
46.
Bourke
VA
,
Watchman
CJ
,
Reith
JD
,
Jorgensen
ML
,
Dieudonnè
A
,
Bolch
WE
.
Spatial gradients of blood vessels and hematopoietic stem and progenitor cells within the marrow cavities of the human skeleton
.
Blood
.
2009
;
114
(
19
):
4077
-
4080
.
47.
Takaku
T
,
Malide
D
,
Chen
J
,
Calado
RT
,
Kajigaya
S
,
Young
NS
.
Hematopoiesis in 3 dimensions: human and murine bone marrow architecture visualized by confocal microscopy
.
Blood
.
2010
;
116
(
15
):
e41
-
e55
.
48.
Flores-Figueroa
E
,
Varma
S
,
Montgomery
K
,
Greenberg
PL
,
Gratzinger
D
.
Distinctive contact between CD34+ hematopoietic progenitors and CXCL12+ CD271+ mesenchymal stromal cells in benign and myelodysplastic bone marrow
.
Lab Invest
.
2012
;
92
(
9
):
1330
-
1341
.
49.
Guezguez
B
,
Campbell
CJV
,
Boyd
AL
, et al
.
Regional localization within the bone marrow influences the functional capacity of human HSCs
.
Cell Stem Cell
.
2013
;
13
(
2
):
175
-
189
.
50.
Aguilar-Navarro
AG
,
Meza-León
B
,
Gratzinger
D
, et al
.
Human aging alters the spatial organization between CD34+ hematopoietic cells and adipocytes in bone marrow
.
Stem Cell Rep
.
2020
;
15
(
2
):
317
-
325
.
51.
Bauer
M
,
Vaxevanis
C
,
Al-Ali
HK
, et al
.
Altered spatial composition of the immune cell repertoire in association to CD34+ blasts in myelodysplastic syndromes and secondary acute myeloid leukemia
.
Cancers
.
2021
;
13
(
2
):
E186
.
52.
Kristensen
HB
,
Andersen
TL
,
Patriarca
A
, et al
.
Human hematopoietic microenvironments
.
PLoS One
.
2021
;
16
(
4
):
e0250081
.
53.
Tjin
G
,
Flores-Figueroa
E
,
Duarte
D
, et al
.
Imaging methods used to study mouse and human HSC niches: current and emerging technologies
.
Bone
.
2019
;
119
:
19
-
35
.
54.
Patel
SS
,
Rodig
SJ
.
Overview of tissue imaging methods
.
Methods Mol Biol
.
2020
;
2055
:
455
-
465
.
55.
Patel
SS
,
Lipschitz
M
,
Pinkus
GS
, et al
.
Multiparametric in situ imaging of NPM1-mutated acute myeloid leukemia reveals prognostically-relevant features of the marrow microenvironment
.
Mod Pathol
.
2020
;
33
(
7
):
1380
-
1388
.
56.
Walters
DK
,
Jelinek
DF
.
Multiplex immunofluorescence of bone marrow core biopsies: visualizing the bone marrow immune contexture
.
J Histochem Cytochem
.
2020
;
68
(
2
):
99
-
112
.
57.
Coutu
DL
,
Kokkaliaris
KD
,
Kunz
L
,
Schroeder
T
.
Multicolor quantitative confocal imaging cytometry
.
Nat Methods
.
2018
;
15
(
1
):
39
-
46
.
58.
Berry
S
,
Giraldo
NA
,
Green
BF
, et al
.
Analysis of multispectral imaging with the AstroPath platform informs efficacy of PD-1 blockade
.
Science
.
2021
;
372
(
6547
):
eaba2609
.
59.
Pan
S
,
Hu
R
,
Long
G
, et al
.
Adversarially regularized graph autoencoder for graph embedding
.
arXiv
.
2018
. 1802.04407.
60.
Patel
SS
,
Weirather
JL
,
Lipschitz
M
, et al
.
The microenvironmental niche in classic Hodgkin lymphoma is enriched for CTLA-4-positive T cells that are PD-1-negative
.
Blood
.
2019
;
134
(
23
):
2059
-
2069
.
61.
Naveiras
O
,
Nardi
V
,
Wenzel
PL
,
Hauschka
PV
,
Fahey
F
,
Daley
GQ
.
Bone-marrow adipocytes as negative regulators of the haematopoietic microenvironment
.
Nature
.
2009
;
460
(
7252
):
259
-
263
.
62.
Lengefeld
J
,
Cheng
C-W
,
Maretich
P
, et al
.
Cell size is a determinant of stem cell potential during aging
.
Sci Adv
.
2021
;
7
(
46
):
eabk0271
.
63.
Heazlewood
SY
,
Ahmad
T
,
Cao
B
, et al
.
High ploidy large cytoplasmic megakaryocytes are hematopoietic stem cells regulators and essential for platelet production
.
Nat Commun
.
2023
;
14
(
1
):
2099
.
64.
Poscablo
DM
,
Worthington
AK
,
Smith-Berdan
S
,
Forsberg
EC
.
Megakaryocyte progenitor cell function is enhanced upon aging despite the functional decline of aged hematopoietic stem cells
.
Stem Cell Rep
.
2021
;
16
(
6
):
1598
-
1613
.
65.
Sanjuan-Pla
A
,
Macaulay
IC
,
Jensen
CT
, et al
.
Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy
.
Nature
.
2013
;
502
(
7470
):
232
-
236
.
66.
Grover
A
,
Sanjuan-Pla
A
,
Thongjuea
S
, et al
.
Single-cell RNA sequencing reveals molecular and functional platelet bias of aged haematopoietic stem cells
.
Nat Commun
.
2016
;
7
:
11075
.
67.
Radtke
AJ
,
Postovalova
E
,
Varlamova
A
, et al
. A Multi-scale, Multiomic Atlas of Human Normal and Follicular Lymphoma Lymph Nodes.
2022
. 2022.06.03.494716.
68.
Pang
WW
,
Price
EA
,
Sahoo
D
, et al
.
Human bone marrow hematopoietic stem cells are increased in frequency and myeloid-biased with age
.
Proc Natl Acad Sci U S A
.
2011
;
108
(
50
):
20012
-
20017
.
69.
Klose
M
,
Florian
MC
,
Gerbaulet
A
,
Geiger
H
,
Glauche
I
.
Hematopoietic stem cell dynamics are regulated by progenitor demand: lessons from a quantitative modeling approach
.
Stem Cell
.
2019
;
37
(
7
):
948
-
957
.
70.
SanMiguel
JM
,
Young
K
,
Trowbridge
JJ
.
Hand in hand: intrinsic and extrinsic drivers of aging and clonal hematopoiesis
.
Exp Hematol
.
2020
;
91
:
1
-
9
.
71.
Young
K
,
Eudy
E
,
Bell
R
, et al
.
Decline in IGF1 in the bone marrow microenvironment initiates hematopoietic stem cell aging
.
Cell Stem Cell
.
2021
;
28
(
8
):
1473
-
1482.e7
.
72.
Kuribayashi
W
,
Oshima
M
,
Itokawa
N
, et al
.
Limited rejuvenation of aged hematopoietic stem cells in young bone marrow niche
.
J Exp Med
.
2021
;
218
(
3
):
e20192283
.
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