1. Stem Cells and Regenerative Medicine
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Murine HSCs contribute actively to native hematopoiesis but with reduced differentiation capacity upon aging

  1. Petter Säwen
  2. Mohamed Eldeeb
  3. Eva Erlandsson
  4. Trine A Kristiansen
  5. Cecilia Laterza
  6. Zaal Kokaia
  7. Göran Karlsson
  8. Joan Yuan
  9. Shamit Soneji
  10. Pankaj K Mandal
  11. Derrick J Rossi
  12. David Bryder  Is a corresponding author
  1. Lund University, Sweden
  2. Harvard University, United States
  3. Boston Children’s Hospital, United States
  4. Gothenburg University, Sweden
Research Article
Cite this article as: eLife 2018;7:e41258 doi: 10.7554/eLife.41258
6 figures, 1 table and 3 additional files

Figures

Figure 1 with 1 supplement
Fgd5-CreERT2 specifically labels HSCs and Fgd5-mediated label progresses throughout the hematopoietic system.

(A) Lineage negative c-kit+ cells (Lin-c-kit+, left), lineage negative c-kit+ Fgd5+ cells (Fgd5+, middle) and lineage negative Fgd5+c-kit+Sca-1+CD150+CD48- cells (HSC-Fgd5+, right) were isolated and subjected to single cell RNA-sequencing. The data was aggregated and visualized in a two-dimensional scatter plot after PCA and tSNE dimensionality reduction. Fgd5+ cells are highlighted in pink (middle), Lin-c-kit+ cells are highlighted in black (left plot) and HSC-Fgd5+ cells are highlighted in blue (right plot). The area that Fgd5+ cells occupy in relation to the transcriptomes of Lin-c-kit+ cells and HSC-Fgd5+ cells is marked by a dotted line (left and right plots). (B) Schematic representation of the Fgd5CreERT2/+; Rosa26lsl-Tomato/+ model. ZsGreen and CreERT2 are expressed from the Fgd5 locus and expression of a Tomato allele is driven by a CAG promoter from the Rosa26 locus and is preceded by a LoxP flanked STOP cassette. (C) Model description; HSCs selectively and continuously express ZsGreen in an Fgd5-dependent manner. Upon Tamoxifen (TAM) administration, HSCs express Tomato and expression of Tomato label is inherited by all progeny of Tomato-expressing HSCs. (D) Representative FACS plots showing Tomato label in BM HSPCs from Fgd5CreERT2/+; Rosa26lsl-Tomato/+ mice that were injected with Tamoxifen 48 hr prior to analysis. (D, lower right) Representative histograms depicting Tomato label in PB cells at various time points after the start of Tamoxifen administration from mice in Figure 3B (T cells 48 weeks, B cells 25 weeks, granulocytes and monocytes 8 weeks, platelets and erythrocytes 13 weeks). Numbers in FACS plots depict the mean % of Tomato labeled cells ± SD (n = 5) and dashed lines in histograms indicates the boundary for Tomato positivity. (E) FACS plots showing H2B-mCherry label retention and Tomato labeling in Lineage-c-kit+CD150+CD48- and Sca1+ or Sca1- cells from a representative mouse that had diluted H2B-mCherry label for 5 weeks and were injected with Tamoxifen 5 days prior to analysis (n = 3; 14–19 weeks old at analysis). (F) The fraction of donor-derived cells among different blood cell lineages was assessed in individual mice 16 weeks post-transplantation in recipients of 5 Tomato+ (n = 8) or 5 Tomato- (n = 7) HSCs. Abbreviations: 2A, 2A self-cleaving peptide; CAG, CAG promoter; loxP, LoxP site.

https://doi.org/10.7554/eLife.41258.003
Figure 1—figure supplement 1
FACS gating strategies for identification of hematopoietic subsets.

(A) The gating scheme for identification of HSCs/MPPs and myeloerythroid progenitors within the LSK and LK populations, respectively. (B) The gating scheme for identification of peripheral blood subsets. Red histogram display Ter119 expression in erythrocytes over an unlabeled control (grey histogram) and platelets identified by gating on scatter characteristics display expression of both CD41 and CD150.

https://doi.org/10.7554/eLife.41258.004
Figure 2 with 2 supplements
Fgd5-CreERT2 mediated lineage tracing reveals robust HSC contribution with distinct kinetics into hematopoietic cell subsets in steady state and after transplantation.

Cohorts of Fgd5CreERT2/+; Rosa26lsl-Tomato/+ mice were chased for up to 83 weeks after receiving one Tamoxifen injection before endpoint analysis of the % of Tomato+ cells in HSCs (A) and hematopoietic progenitor and PB cell subsets (B). The fraction of Tomato+ cells in each indicated hematopoietic cell subset was divided by the % Tomato label in HSCs in corresponding mice to determine the ratio of Tomato+ cells relative to HSCs in individual mice. The average ratio is plotted according to time after the Tamoxifen (TAM) injection and displayed in a heat map format. Mice were analyzed at 1–4 days (n = 18) and at 4 (n = 7), 8 (n = 8), 12 (n = 8), 16 (n = 4), 28 (n = 4), 32 (n = 5) and 83 (n = 3) weeks after the Tamoxifen injection. All mice were between 5 and 11 weeks old at the time of Tamoxifen injection. (C) PCA plot of multiplexed qRT-PCR data for 48 genes from triplicates of 10 cells from each of the indicated populations. Ellipses show an area where a new observation from the same group would position itself with a probability of 0.95. Numbers indicate the % of variance in the total data set that the respective PC explains. (D) For individual animals, the % of Tomato+ cells in PB cell types was divided by the % of Tomato+ cells in the indicated progenitor cell types and this value was plotted according to the duration of the chase period. Symbols represent individual mice (number of mice analyzed as in A and B). A dashed line is drawn to indicate an equilibrium-ratio of 1. (E) Ratios calculated as in B and plotted according to the number of weeks after transplantation or after the start of Tamoxifen administration for steady state/native mice (n = 3 for WBM tx in CD45 depleted, n = 5 for WBM tx in irradiated, n = 11 for HSC tx in irradiated, n = 4–8 for 1x TAM, n = 4–9 for continous TAM). All Fgd5CreERT2/+; Rosa26lsl-Tomato/+ mice/cells were between 5 and 11 weeks old at the time of Tamoxifen administration or when used as cell donors for transplantation.

https://doi.org/10.7554/eLife.41258.005
Figure 2—figure supplement 1
Label induction and progression in 1x Tamoxifen treated adult Fgd5CreERT2/+; Rosa26lsl-Tomato/+ mice.

Left column: percentages of Tomato+ cells among the indicated hematopoietic subsets in bone marrow and peripheral blood at the indicated time after a single Tamoxifen injection. Right column: ratio of the % of Tomato+ cells in the indicated hematopoietic cell subsets relative to the % of Tomato+ HSCs in corresponding mice. Mice were analyzed 24 hr (n = 6), 48 hr (n = 6), 96 hr (n = 6), 4 w (n = 7), 8 w (n = 8), 12 w (n = 8), 16 w (n = 4), 28 w (n = 4), 32 w (n = 5) and 83 w (n = 3) after Tamoxifen injection. Cohorts of mice were injected with Tamoxifen in five different experiments. Symbols indicate individual mice and bars indicate the mean Tomato level at each time point evaluated.

https://doi.org/10.7554/eLife.41258.006
Figure 2—figure supplement 2
In vitro differentiation potential of LSK HSPCs.

Single GMLPs (n = 279), HSCs (n = 240) and LSKCD150+CD48+ cells (n = 460) were seeded into Terasaki wells and cultured for 6 days before scoring wells for erythroid cells and megakaryocytes by microscopic evaluation. The % of wells containing erythroid cells (red), megakaryocytes (orange) or cells other than erythroid or megakaryocytes (green) are plotted in filled bars from the different indicated cells evaluated.

https://doi.org/10.7554/eLife.41258.007
Fgd5-mediated lineage tracing after complete HSC labeling reveals limited adult HSCs contribution to tissue-resident immune cell subsets.

(A) H2B-mCherry label retention in HSCs and myeloid progenitors after 5 weeks of chase in mice continuously fed Tamoxifen (TAM) containing food (blue histograms, n = 7) or normal food (red histograms, n = 6) during the chase period. Green histograms depict an unlabeled control. Dashed lines indicate the boundary for H2B-mCherry positivity. Lineage negative c-kit+Sca1- cells are denoted as myeloid progenitors. (B) Cohorts of Fgd5CreERT2/+; Rosa26lsl-Tomato/+ mice were continuously fed Tamoxifen food for 16 weeks (TAM phase) and thereafter normal chow during a chase phase of 32 weeks (n = 5) or 41 weeks (n = 4). During the TAM and chase phases, PB was regularly analyzed for Tomato label in the indicated subsets. Data points indicate the average % of Tomato label in each indicated subset. (C–E) Endpoint analysis, after 41 weeks chase, in mice from Figure 3B (n = 3). (C) Representative FACS plots showing the gating strategy to identify HSCs in lineage negative BM cells (left, middle) and depiction of the Tomato label in HSCs (right, red histogram) compared to an unlabeled control (blue histogram). Dashed line indicates the boundary for Tomato positivity, arrows indicate the gating strategy. (D) Representative FACS plots of cells isolated from skin tissue (epidermis) or the peritoneal cavity. Histograms show Tomato label (bottom left and right) or ZsGreen label (bottom middle) in the indicated subsets. Dashed line indicate the boundary for Tomato positivity, arrows indicate the gating hierarchy, gates and corresponding histograms are color matched. (E) Representative confocal images of the brain. (Left) IBA1 positive cells (green) are not labeled with Tomato (red, middle) while blood vessels are labeled with Tomato (middle). (Right) Merged images display both IBA1 staining and Tomato label. Scale bar = 50 μm. Mice in B-E were 5–6 weeks old at the start of Tamoxifen administration.

https://doi.org/10.7554/eLife.41258.008
Figure 4 with 1 supplement
Fgd5 mediate lineage tracing reveals gradually declining HSC contribution to hematopoiesis with age.

(A) Cohorts of aged (11–12 months; n = 3) or young (23–25 days; n = 5,) Fgd5CreERT2/+; Rosa26lsl-Tomato/+ mice were injected with Tamoxifen 5 times on consecutive days and analyzed for Tomato label in LSK-HSPCs on day 6. Bars indicate median %. (B) Fgd5CreERT2/+; Rosa26lsl-Tomato/+ mice were injected with Tamoxifen for 5 consecutive days and analyzed for Tomato label in LSK-HSPCs 21 days later. The % of Tomato+ cells in MPP2-4 was divided by the % of Tomato+ HSCs in corresponding mice to determine the ratio of Tomato+ cells relative to HSCs. Ratios are plotted according to the age of mice at the time of Tamoxifen injection. (C) Indicated LSK subsets were isolated from Fgd5CreERT2/+; Rosa26lsl-Tomato/+ mice and transplanted into WT mice on continuous Tamoxifen. Multilineage hematopoiesis and Tomato contribution was assed in PB 4, 8, 12 and 16 weeks after transplantation. Values in parenthesis indicate the number of mice with Tomato+ offspring. (D) Young (29 days; n = 3) and old (16 months; n = 4) Fgd5CreERT2/+; Rosa26lsl-Tomato/+ mice were injected once with Tamoxifen and analyzed for Tomato label 18 weeks later. The % of Tomato+ cells in each indicated cell type was divided by the % of Tomato+ HSCs in corresponding mice to determine the ratio of Tomato+ cells relative to HSCs. (E) Model depicting HSC contribution to native hematopoiesis. Arrow weights and arrow lengths indicate the magnitude and the kinetics of HSC contribution to the indicated cell type respectively. The declining HSC contribution to hematopoiesis with age can be traced to a reduced differentiation of HSCs to MPPs (red arrows).

https://doi.org/10.7554/eLife.41258.009
Figure 4—figure supplement 1
Aging associates with reduced multilineage HSC contribution.

The percentages of Tomato+ cells among the indicated hematopoietic cells in bone marrow and peripheral blood 18 weeks following injection of Tamoxifen.

https://doi.org/10.7554/eLife.41258.010
Author response image 1
The Tomato fluorescent protein does not influence on the in vivo differentiation potential of HSCs.
Author response image 2
The expression of Fgd5 in HSC subsets of young and old mice.

Tables

Key resources table
Reagent type
(species)
or resource
DesignationSource or
reference
IdentifiersAdditional
information
Strain, strain
background
(M. musculus)
Fgd5-CreERT2PMID:24958848RRID:IMSR_JAX:027789
Strain, strain
background
(M. musculus)
Rosa26-rtTA;
Col1a1-tetO-
H2B-mCherry
PMID:17554301RRID:IMSR_JAX:014602
Strain, strain
background
(M. musculus)
Rosa26-
Lox-Stop-
Lox-Tomato
PMID:20023653RRID:IMSR_JAX:007905
AntibodyB220 PECy5BiolegendRRID:AB_312994(1:400)
Clone: RA3-6B2
AntibodyB220 biotinBiolegendRRID:AB_312989(1:200)
Clone: RA3-6B2
AntibodyB220 APCBiolegendRRID:AB_312997(1:400)
Clone: RA3-6B2
AntibodyCD105 PECy7BiolegendRRID:AB_1027700(1:200)
Clone: MJ7/18
AntibodyCD115 BV605BiolegendRRID:AB_2562760(1:200)
Clone: CSF-1R
AntibodyCD11b PECy5BiolegendRRID:AB_312793(1:400)
Clone: M1/70
AntibodyCD11b biotinBiolegendRRID:AB_312787(1:200)
Clone: M1/70
AntibodyCD11b APCBiolegendRRID:AB_312795(1:800)
Clone: M1/70
AntibodyCD11b APC-Cy7BiolegendRRID:AB_830641(1:200)
Clone: M1/70
AntibodyCD11c BV570BiolegendRRID:AB_10900261(1:200)
Clone: N418
AntibodyCD150 APCBiolegendRRID:AB_493460(1:400)
Clone:
TC15-12F12.2
AntibodyCD150 PEBiolegendRRID:AB_313683(1:200)
Clone:
TC15-12F12.2
AntibodyCD16/32 AL700eBioscienceRRID:AB_493994(1:100)
Clone: 93
AntibodyCD19 PECy7eBioscienceRRID:AB_657663(1:200)
Clone: 1D3
AntibodyCD19 BV-786BD-HorizonRRID:AB_2738141(1:200)
Clone: 1D3
AntibodyCD25 APCeBioscienceRRID:AB_469366(1:100)
Clone: PC 61.5
AntibodyCD31 PerCpCy5.5BiolegendRRID:AB_2566761(1:200)
Clone: MEC13.3
AntibodyCD3e PECy5BiolegendRRID:AB_312675(1:400)
Clone:
145–2 C11
AntibodyCD3e biotinBiolegendRRID:AB_2563947(1:200)
Clone: 17A2
AntibodyCD3 AL700BiolegendRRID:AB_493697(1:200)
Clone: 17A2
AntibodyCD4 BV711BiolegendRRID:AB_2562607(1:200)
Clone: RM4-5
AntibodyCD4 APC eFl780eBioscienceRRID:AB_1272219(1:200)
Clone: RM4-5
AntibodyCD41 PerCP-eFl710eBioscienceRRID:AB_10855042(1:200)
Clone: MWReg30
AntibodyCD45.1 AL700BiolegendRRID:AB_493733(1:200)
Clone: A20
AntibodyCD45.2 PECy7BiolegendRRID:AB_1186098(1:200)
Clone: 104
AntibodyCD45.2 PE/
Dazzle
BiolegendRRID:AB_2564177(1:200)
Clone: 104
AntibodyCD45.2 BV785SonyRRID:AB_2562604(1:200)
Clone: 104
AntibodyCD45.2 biotinBiolegendRRID:AB_313441(1:200)
Clone: 104
AntibodyCD48 PECy7BiolegendRRID:AB_2075049(1:200)
Clone:
HM48-1
AntibodyCD48 AL700BiolegendRRID:AB_10612755(1:200)
Clone:
HM48-1
AntibodyCD5 BV-421BD-HorizonRRID:AB_2737758(1:200)
Clone: 53–7.3
AntibodyCD8 PerCpCy5.5SonyRRID:AB_2075239(1:200)
Clone: 53–6.7
Antibodyc-kit APCeFl780eBioscienceRRID:AB_1272177(1:200)
Clone: 2B8
Antibodyc-kit APCBiolegendRRID:AB_313221(1:100)
Clone: 2B8
AntibodyF4/80 BV421BiolegendRRID:AB_11203717(1:200)
Clone: BM8
AntibodyFlt3 biotineBioscienceRRID:AB_466600(1:200)
Clone: AZF10
AntibodyGr1 PECy5BiolegendRRID:AB_313375(1:400)
Clone: RB6-8C5
AntibodyGr1 FITCBD PHRRID:AB_394643(1:400)
Clone: RB6-8C5
AntibodyGr1 biotinBiolegendRRID:AB_313369(1:200)
Clone: RB6-8C5
AntibodyGr1 BV711SonyRRID:AB_2562549(1:200)
Clone: RB6-8C5
AntibodyIL7Ra BV510SonyRRID:AB_2564576(1:200)
Clone: A7R34
AntibodyLy6G APC/Fire750BiolegendRRID:AB_2616733(1:200)
Clone: 1A8
AntibodyNK1.1 PECy5BiolegendRRID:AB_493591(1:400)
Clone: PK136
AntibodyNK1.1 Pacific BlueBiolegendRRID:AB_2132712(1:200)
Clone: PK136
AntibodySca1 Pacific BlueBiolegendRRID:AB_2143237(1:200)
Clone: E13-161.7
AntibodyTcR Vγ3 APCBiolegendRRID:AB_10895900(1:200)
Clone: 536
AntibodyTcR γ/δ BV605BiolegendRRID:AB_2563356(1:200)
Clone: GL3
AntibodyTer119 PECy5BiolegendRRID:AB_313711(1:400)
Clone: Ter-119
AntibodyTer119 biotinBiolegendRRID:AB_313705(1:200)
Clone: Ter-119
AntibodyTer119 PerCpCy5.5BiolegendRRID:AB_893636(1:200)
Clone: Ter-119
Peptide,
recombinant
protein
Streptavidin
BV605
Biolegend(1:400)
Peptide,
recombinant
protein
Streptavidin-
Saporin
PMID:
27272386
Advanced
Targeting
Systems
Chemical
compound,
drug
TamoxifenSigma-
Aldrich
I.p. 50 mg/kg
Chemical
compound,
drug
DoxycyclineSsniff
Spezialdiäten
Food 2 g/kg
Chemical
compound,
drug
TamoxifenSsniff
Spezialdiäten
Food
400 mg/kg
Tamoxifen
Citrate
Software,
algorithm
FlowjoFlowJo
(https://www.
flowjo.com/solutions/flowjo)
RRID:S
CR_008520
Software,
algorithm
Microsoft
Excel
Microsoft
Excel
(https://www.microsoft.com/en-gb/)
RRID:
SCR_016137
Software,
algorithm
Graphpad
Prism
GraphPad
Prism (https://graphpad.com)
RRID:
SCR_002798

Additional files

Supplementary file 1

Definitions of cells used throughout study.

https://doi.org/10.7554/eLife.41258.011
Supplementary file 2

List of analyzed genes in mutiplexed qRT-PCR gene expression analyses (Figure 2C).

https://doi.org/10.7554/eLife.41258.012
Transparent reporting form
https://doi.org/10.7554/eLife.41258.013

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