Nature - USA (2020-02-13)

Nature | Vol 578 | 13 February 2020 | 279

mice confirmed the complete absence of GFP expression in any mature
lineage-positive (LIN+) haematopoietic cells (Extended Data Fig. 2a,
Supplementary File 2). GFP expression was predominantly restricted to
a small fraction of CKIT+SCA1+ cells (Fig. 1a). Using standard phenotypi-
cal parameters^12 ,^13 , we found that 28.83 ± 11.99% (mean ± s.d.) of bone
marrow cells gated solely on GFP could be categorized as LT-HSCs,
26.61 ± 9.86% as short-term HSCs (ST-HSCs) and 30.12 ± 12.8% as MPPs
(Fig. 1a, Supplementary File 1). Within the phenotypic LT-HSC compart-
ment, approximately 60% of cells expressed GFP, compared to 6% of
MPPs (Fig. 1b). We refer to this mixed population as haematopoietic
stem and progenitor cells (HSPCs). Notably, GFP was not expressed in
non-haematopoietic compartments of the bone marrow (Extended
Data Fig. 2b, c, Supplementary File 3).
With the aim of eliminating the labelling of MPPs in the Mds1GFP/+
model, we reasoned that the additional expression of a gene associ-
ated with early differentiation could facilitate exclusive identifica-
tion of LT-HSCs. Increased brightness of the reporter in phenotyical
LT-HSCs was inversely correlated with the expression of Flt3, a gene
whose expression has been associated with the loss of long-term self-
renewal^14 ,^15 (Extended Data Fig. 2d). Taking advantage of the fact that
the GFP coding sequences in the Mds1GFP/+ allele are flanked by loxP sites
(Extended Data Fig. 1a), we introduced a Flt3Cre allele into our model
(Extended Data Fig. 3a). This allele drives Cre-mediated recombina-
tion in cells, beginning in the ST-HSC compartment^14 ,^15 (Extended Data

Fig. 3b). Characterization of Mds1GFP/+Flt3Cre mice revealed an extremely

rare GFP+ population (referred to as MFG cells) that corresponds to

only 0.022 ± 0.013% of the lineage-negative bone marrow (Fig. 1a, b).

Remarkably, approximately 85% of cells gated solely on the basis of GFP

resided in the phenotypically defined LT-HSC fraction (Fig. 1a, Extended

Data Fig. 3e). Another 10% of MFG GFP+ cells displayed slightly lower

levels of CD150 and might be classified as ST-HSCs (Fig. 1a), and the

other 5% represents CD150+CD48− cells that express lower levels of SCA1

and are likely to be megakaryocyte progenitors (MkPs; Extended Data

Fig. 3c, Supplementary File 1). MFG cells constituted only about 12% of

the phenotypical LT-HSC population (Fig. 1b). The specificity of LT-HSC

labelling in MFG mice was recapitulated in bone marrow from multi-

ple locations (Extended Data Fig. 3d). MFG cells represented a largely

quiescent population (Fig. 1c, Extended Data Fig. 3g) that express rela-

tively high levels of SCA1 and EPCR and little or no CD34 (Extended

Data Fig. 3e, f ), consistent with previously described dormant HSCs^16.

To further validate our combined Mds1GFP/+ Flt3Cre model, we per-

formed single-cell RNA sequencing (scRNA-seq) in cells isolated

exclusively on the basis of GFP expression^17. The resulting transcrip-

tomes were then extrapolated to a published single-cell transcrip-

tional map of LT-HSCs, ST-HSCs, and multiple MPP populations

(MPP2/3/4)^18. Strikingly, virtually all MFG+ transcriptomes mapped

to the most unprimed cluster of cells, in which phenotypic LT-HSCs

also reside (Fig. 1d, Extended Data Fig. 4a). A small fraction of MFG+

ST-HSC

MFG GFP LT-HSC

Mk

Erythroid

Monocyte

Basophil

B cell

Monocyte

Dendriticcell

T cell

e

c SLAM MFG

DAPI

Ki67

d

b

MDS1GFP

Mds1+/+ Mds1GFP/+Flt3Cre

MPP3/4 ST-HSC LT-HSC

Mds1GFP/+

GFP+ GFP+

0% 0.98 ± 0.82%

11.79 ± 5.89%

GFP+

6.014 ± 4.76% 38.46 ± 22.25%

59.93 ± 18.18%

a Lin– fraction

C-KIT

GFP

C-KIT

SCA-1

CD48

CD150

Mds1+/+Flt3Cre

Mds1GFP/+

Mds1GFP/+Flt3Cre

0.022 ±

0.013

0.48 ±

0.44

88.53 ± 5.95%

89.16 ± 2.675%

GFPGFP+

• 
  

0%

10.92 ± 6.76%

85.21 ±

8.03%

26.61 ± 9.86%

28.83 ±

11.99%

30.12 ± 12.8%

Overall chimaerism

0.1

1

10

100

0.1

1

10

100

0123 4

Granulocyte chimaerism

5

0123 4 5

Neutrophil

SLAM

MFG

50.6GO

35.2G1

2.59SG2–M3.93 G2–M 0

G1 0

85.7G0

S 0

Months after transplant

Donor contribution (%)

Fig. 1 | Generation and characterization of Mds1GFP/+Flt3Cre (MFG) mice.
a, b, Flow cytometric analysis of Mds1GFP/+ only mice (n = 10) and Mds1GFP/+Flt3Cre
mice (n = 13); mean ± s.d. c, Cell cycle analysis of GFP+ cells from MFG
mice versus HSCs isolated by LIN−SCA-1+C-KIT+CD150+CD48− (SLAM)
immunophenotype. Representative analysis shown, depicting data from
multiple mice (MFG, n = 7; SLAM, n = 2) that were pooled together to acquire the
displayed data. d, SPRING plot layout of transcriptomes of 50 single MFG+ HSCs

projected in published scRNA dataset of HSCs and MPPs^18. Blue, LT-HSCs

(n = 789); red, ST-HCSs (n = 742); grey, other cells; bright green, MGF HSCs

(n = 46). Each dot represents one cell. e, Overall and granulocyte chimaerism

after transplantation in primary lethally irradiated recipients transplanted

with 25 MFG+ (n = 6 mice) or SLAM cells (n = 5 mice) from Mds1GFP/+Flt3Cre mice.

Each line represents an individual mouse. Only engrafted mice are shown.