Key Points

  • FL-HSCs mainly use oxidative phosphorylation but with normal glycolysis, as indicated by a highly responsive NADH/NAD+ sensor.

  • FL-HSC activities are tightly regulated by the STAT3/MDH1-mediated malate-aspartate NADH shuttle.

Abstract

The connections between energy metabolism and stemness of hematopoietic stem cells (HSCs) at different developmental stages remain largely unknown. We generated a transgenic mouse line for the genetically encoded NADH/NAD+ sensor (SoNar) and demonstrate that there are 3 distinct fetal liver hematopoietic cell populations according to the ratios of SoNar fluorescence. SoNar-low cells had an enhanced level of mitochondrial respiration but a glycolytic level similar to that of SoNar-high cells. Interestingly, 10% of SoNar-low cells were enriched for 65% of total immunophenotypic fetal liver HSCs (FL-HSCs) and contained approximately fivefold more functional HSCs than their SoNar-high counterparts. SoNar was able to monitor sensitively the dynamic changes of energy metabolism in HSCs both in vitro and in vivo. Mechanistically, STAT3 transactivated MDH1 to sustain the malate-aspartate NADH shuttle activity and HSC self-renewal and differentiation. We reveal an unexpected metabolic program of FL-HSCs and provide a powerful genetic tool for metabolic studies of HSCs or other types of stem cells.

REFERENCES

REFERENCES
1.
Boisset
JC
,
van Cappellen
W
,
Andrieu-Soler
C
,
Galjart
N
,
Dzierzak
E
,
Robin
C
.
In vivo imaging of haematopoietic cells emerging from the mouse aortic endothelium
.
Nature
.
2010
;
464
(
7285
):
116
-
120
.
2.
Ema
H
,
Nakauchi
H
.
Expansion of hematopoietic stem cells in the developing liver of a mouse embryo
.
Blood
.
2000
;
95
(
7
):
2284
-
2288
.
3.
Khan
JA
,
Mendelson
A
,
Kunisaki
Y
, et al
.
Fetal liver hematopoietic stem cell niches associate with portal vessels
.
Science
.
2016
;
351
(
6269
):
176
-
180
.
4.
Wilson
A
,
Laurenti
E
,
Oser
G
, et al
.
Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair [published correction appears in Cell. 2009;138(1):209]
.
Cell
.
2008
;
135
(
6
):
1118
-
1129
.
5.
Seita
J
,
Weissman
IL
.
Hematopoietic stem cell: self-renewal versus differentiation
.
Wiley Interdiscip Rev Syst Biol Med
.
2010
;
2
(
6
):
640
-
653
.
6.
Graf
T
,
Enver
T
.
Forcing cells to change lineages
.
Nature
.
2009
;
462
(
7273
):
587
-
594
.
7.
Miharada
K
,
Karlsson
G
,
Rehn
M
, et al
.
Cripto regulates hematopoietic stem cells as a hypoxic-niche-related factor through cell surface receptor GRP78
.
Cell Stem Cell
.
2011
;
9
(
4
):
330
-
344
.
8.
Nakada
D
,
Saunders
TL
,
Morrison
SJ
.
Lkb1 regulates cell cycle and energy metabolism in haematopoietic stem cells
.
Nature
.
2010
;
468
(
7324
):
653
-
658
.
9.
Ito
K
,
Carracedo
A
,
Weiss
D
, et al
.
A PML–PPAR-δ pathway for fatty acid oxidation regulates hematopoietic stem cell maintenance
.
Nat Med
.
2012
;
18
(
9
):
1350
-
1358
.
10.
Eliasson
P
,
Jönsson
JI
.
The hematopoietic stem cell niche: low in oxygen but a nice place to be
.
J Cell Physiol
.
2010
;
222
(
1
):
17
-
22
.
11.
Chow
DC
,
Wenning
LA
,
Miller
WM
,
Papoutsakis
ET
.
Modeling pO(2) distributions in the bone marrow hematopoietic compartment. II. Modified Kroghian models
.
Biophys J
.
2001
;
81
(
2
):
685
-
696
.
12.
Takubo
K
,
Goda
N
,
Yamada
W
, et al
.
Regulation of the HIF-1alpha level is essential for hematopoietic stem cells
.
Cell Stem Cell
.
2010
;
7
(
3
):
391
-
402
.
13.
Laurenti
E
,
Varnum-Finney
B
,
Wilson
A
, et al
.
Hematopoietic stem cell function and survival depend on c-Myc and N-Myc activity
.
Cell Stem Cell
.
2008
;
3
(
6
):
611
-
624
.
14.
Takubo
K
,
Nagamatsu
G
,
Kobayashi
CI
, et al
.
Regulation of glycolysis by Pdk functions as a metabolic checkpoint for cell cycle quiescence in hematopoietic stem cells
.
Cell Stem Cell
.
2013
;
12
(
1
):
49
-
61
.
15.
Qian
P
,
He
XC
,
Paulson
A
, et al
.
The Dlk1-Gtl2 locus preserves LT-HSC function by inhibiting the PI3K-mTOR pathway to restrict mitochondrial metabolism
.
Cell Stem Cell
.
2016
;
18
(
2
):
214
-
228
.
16.
Cabezas-Wallscheid
N
,
Buettner
F
,
Sommerkamp
P
, et al
.
Vitamin A-retinoic acid signaling regulates hematopoietic stem cell dormancy
.
Cell
.
2017
;
169
(
5
):
807
-
823.e19
.
17.
Simsek
T
,
Kocabas
F
,
Zheng
J
, et al
.
The distinct metabolic profile of hematopoietic stem cells reflects their location in a hypoxic niche
.
Cell Stem Cell
.
2010
;
7
(
3
):
380
-
390
.
18.
Kocabas
F
,
Xie
L
,
Xie
J
, et al
.
Hypoxic metabolism in human hematopoietic stem cells
.
Cell Biosci
.
2015
;
5
:
39
.
19.
Kocabas
F
,
Zheng
J
,
Thet
S
, et al
.
Meis1 regulates the metabolic phenotype and oxidant defense of hematopoietic stem cells
.
Blood
.
2012
;
120
(
25
):
4963
-
4972
.
20.
de Almeida
MJ
,
Luchsinger
LL
,
Corrigan
DJ
, et al
.
Dye-independent methods reveal elevated mitochondrial mass in hematopoietic stem cells
.
Cell Stem Cell
.
2017
;
21
(
6
):
725
-
729.e4
.
21.
Rimmelé
P
,
Liang
R
,
Bigarella
CL
, et al
.
Mitochondrial metabolism in hematopoietic stem cells requires functional FOXO3
.
EMBO Rep
.
2015
;
16
(
9
):
1164
-
1176
.
22.
Ansó
E
,
Weinberg
SE
,
Diebold
LP
, et al
.
The mitochondrial respiratory chain is essential for haematopoietic stem cell function
.
Nat Cell Biol
.
2017
;
19
(
6
):
614
-
625
.
23.
Manesia
JK
,
Xu
Z
,
Broekaert
D
, et al
.
Highly proliferative primitive fetal liver hematopoietic stem cells are fueled by oxidative metabolic pathways
.
Stem Cell Res (Amst)
.
2015
;
15
(
3
):
715
-
721
.
24.
Wang
YH
,
Israelsen
WJ
,
Lee
D
, et al
.
Cell-state-specific metabolic dependency in hematopoiesis and leukemogenesis
.
Cell
.
2014
;
158
(
6
):
1309
-
1323
.
25.
Agathocleous
M
,
Meacham
CE
,
Burgess
RJ
, et al
.
Ascorbate regulates haematopoietic stem cell function and leukaemogenesis
.
Nature
.
2017
;
549
(
7673
):
476
-
481
.
26.
Zhao
Y
,
Hu
Q
,
Cheng
F
, et al
.
SoNar, a highly responsive NAD+/NADH sensor, allows high-throughput metabolic screening of anti-tumor agents
.
Cell Metab
.
2015
;
21
(
5
):
777
-
789
.
27.
Patterson
GH
,
Knobel
SM
,
Arkhammar
P
,
Thastrup
O
,
Piston
DW
.
Separation of the glucose-stimulated cytoplasmic and mitochondrial NAD(P)H responses in pancreatic islet beta cells
.
Proc Natl Acad Sci USA
.
2000
;
97
(
10
):
5203
-
5207
.
28.
Yamada
K
,
Hara
N
,
Shibata
T
,
Osago
H
,
Tsuchiya
M
.
The simultaneous measurement of nicotinamide adenine dinucleotide and related compounds by liquid chromatography/electrospray ionization tandem mass spectrometry
.
Anal Biochem
.
2006
;
352
(
2
):
282
-
285
.
29.
Yang
H
,
Yang
T
,
Baur
JA
, et al
.
Nutrient-sensitive mitochondrial NAD+ levels dictate cell survival
.
Cell
.
2007
;
130
(
6
):
1095
-
1107
.
30.
Yu
Q
,
Heikal
AA
.
Two-photon autofluorescence dynamics imaging reveals sensitivity of intracellular NADH concentration and conformation to cell physiology at the single-cell level
.
J Photochem Photobiol B
.
2009
;
95
(
1
):
46
-
57
.
31.
Zhao
Y
,
Wang
A
,
Zou
Y
,
Su
N
,
Loscalzo
J
,
Yang
Y
.
In vivo monitoring of cellular energy metabolism using SoNar, a highly responsive sensor for NAD(+)/NADH redox state
.
Nat Protoc
.
2016
;
11
(
8
):
1345
-
1359
.
32.
Pomorski
A
,
Kochańczyk
T
,
Miłoch
A
,
Krężel
A
.
Method for accurate determination of dissociation constants of optical ratiometric systems: chemical probes, genetically encoded sensors, and interacting molecules
.
Anal Chem
.
2013
;
85
(
23
):
11479
-
11486
.
33.
Bolbat
A
,
Schultz
C
.
Recent developments of genetically encoded optical sensors for cell biology
.
Biol Cell
.
2017
;
109
(
1
):
1
-
23
.
34.
Zhao
Y
,
Yang
Y
.
Real-time and high-throughput analysis of mitochondrial metabolic states in living cells using genetically encoded NAD+/NADH sensors
.
Free Radic Biol Med
.
2016
;
100
:
43
-
52
.
35.
Zhao
Y
,
Jin
J
,
Hu
Q
, et al
.
Genetically encoded fluorescent sensors for intracellular NADH detection
.
Cell Metab
.
2011
;
14
(
4
):
555
-
566
.
36.
Gu
H
,
Chen
C
,
Hao
X
, et al
.
Sorting protein VPS33B regulates exosomal autocrine signaling to mediate hematopoiesis and leukemogenesis
.
J Clin Invest
.
2016
;
126
(
12
):
4537
-
4553
.
37.
Redell
MS
,
Ruiz
MJ
,
Alonzo
TA
,
Gerbing
RB
,
Tweardy
DJ
.
Stat3 signaling in acute myeloid leukemia: ligand-dependent and -independent activation and induction of apoptosis by a novel small-molecule Stat3 inhibitor
.
Blood
.
2011
;
117
(
21
):
5701
-
5709
.
38.
Stein
LR
,
Imai
S
.
The dynamic regulation of NAD metabolism in mitochondria
.
Trends Endocrinol Metab
.
2012
;
23
(
9
):
420
-
428
.
39.
Veech
RL
,
Eggleston
LV
,
Krebs
HA
.
The redox state of free nicotinamide-adenine dinucleotide phosphate in the cytoplasm of rat liver
.
Biochem J
.
1969
;
115
(
4
):
609
-
619
.
40.
Ido
Y
.
Pyridine nucleotide redox abnormalities in diabetes
.
Antioxid Redox Signal
.
2007
;
9
(
7
):
931
-
942
.
41.
Hao
X
,
Gu
H
,
Chen
C
, et al
.
Metabolic imaging reveals a unique preference of symmetric cell division and homing of leukemia-initiating cells in an endosteal niche
.
Cell Metab
.
2019
;
29
(
4
):
950
-
965.e6
.
42.
Abbrescia
DI
,
La Piana
G
,
Lofrumento
NE
.
Malate-aspartate shuttle and exogenous NADH/cytochrome c electron transport pathway as two independent cytosolic reducing equivalent transfer systems
.
Arch Biochem Biophys
.
2012
;
518
(
2
):
157
-
163
.
43.
Yang
H
,
Zhou
L
,
Shi
Q
, et al
.
SIRT3-dependent GOT2 acetylation status affects the malate-aspartate NADH shuttle activity and pancreatic tumor growth
.
EMBO J
.
2015
;
34
(
8
):
1110
-
1125
.
44.
Oh
IH
,
Eaves
CJ
.
Overexpression of a dominant negative form of STAT3 selectively impairs hematopoietic stem cell activity
.
Oncogene
.
2002
;
21
(
31
):
4778
-
4787
.
45.
Chung
YJ
,
Park
BB
,
Kang
YJ
,
Kim
TM
,
Eaves
CJ
,
Oh
IH
.
Unique effects of Stat3 on the early phase of hematopoietic stem cell regeneration
.
Blood
.
2006
;
108
(
4
):
1208
-
1215
.
46.
Darnell
JE
Jr.
,
Kerr
IM
,
Stark
GR
.
Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins
.
Science
.
1994
;
264
(
5164
):
1415
-
1421
.
47.
Hu
M
,
Zeng
H
,
Chen
S
, et al
.
SRC-3 is involved in maintaining hematopoietic stem cell quiescence by regulation of mitochondrial metabolism in mice
.
Blood
.
2018
;
132
(
9
):
911
-
923
.
48.
Zhou
F
,
Li
X
,
Wang
W
, et al
.
Tracing haematopoietic stem cell formation at single-cell resolution
.
Nature
.
2016
;
533
(
7604
):
487
-
492
.
49.
Jassinskaja
M
,
Johansson
E
,
Kristiansen
TA
, et al
.
Comprehensive proteomic characterization of ontogenic changes in hematopoietic stem and progenitor cells
.
Cell Rep
.
2017
;
21
(
11
):
3285
-
3297
.
50.
Ding
L
,
Saunders
TL
,
Enikolopov
G
,
Morrison
SJ
.
Endothelial and perivascular cells maintain haematopoietic stem cells
.
Nature
.
2012
;
481
(
7382
):
457
-
462
.
51.
Ding
L
,
Morrison
SJ
.
Haematopoietic stem cells and early lymphoid progenitors occupy distinct bone marrow niches [published correction appears in Nature. 2014;514(7521):262]
.
Nature
.
2013
;
495
(
7440
):
231
-
235
.
52.
Tothova
Z
,
Kollipara
R
,
Huntly
BJ
, et al
.
FoxOs are critical mediators of hematopoietic stem cell resistance to physiologic oxidative stress
.
Cell
.
2007
;
128
(
2
):
325
-
339
.
53.
Gan
B
,
Hu
J
,
Jiang
S
, et al
.
Lkb1 regulates quiescence and metabolic homeostasis of haematopoietic stem cells
.
Nature
.
2010
;
468
(
7324
):
701
-
704
.
54.
Luchsinger
LL
,
de Almeida
MJ
,
Corrigan
DJ
,
Mumau
M
,
Snoeck
HW
.
Mitofusin 2 maintains haematopoietic stem cells with extensive lymphoid potential
.
Nature
.
2016
;
529
(
7587
):
528
-
531
.
55.
Tao
R
,
Zhao
Y
,
Chu
H
, et al
.
Genetically encoded fluorescent sensors reveal dynamic regulation of NADPH metabolism
.
Nat Methods
.
2017
;
14
(
7
):
720
-
728
.
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