Stem Cell Reports, Volume 1
Supplemental Information
Transcriptome Analysis Identifies Regulators of Hematopoietic Stem and Progenitor
Cells
Roi Gazit, Brian S. Garrison, Tata Nageswara Rao, Tal Shay, James Costello, Jeff Ericson,
Francis Kim, James J. Collins, Aviv Regev, Amy J. Wagers, Derrick J. Rossi, and The
Immunological Genome Project Consortium
Inventory of Supplementary Information
Supplementary Figures
Accompanies Results Section: HSPCs are transcriptionally enriched for genes associated with
transit amplification.
Supplementary Figure 1: Enriched genes within hematopoietic stem and progenitor cells.
(a) Heat map of 1605 HSPC-enriched genes identified through ANOVA.
(b) Graph showing enrichment of nc-RNA and cell cycle gene groups in B cell progenitors,
consistent with their proliferative potential.
(c) Graph showing expression nc-RNA and cell cycle gene groups in T cell progenitors,
consistent with their proliferative potential.
Accompanies Results Section: Identification of a novel group of CH3 zinc finger KRAB domain
containing transcriptional repressors in multi-potent stem and progenitor cells
Supplementary Figure 2: Enriched genes within multipotent stem and progenitor cells.
Heat map representing 443 multipotent cell-enriched genes using ANOVA.
Supplementary Figure 3: Expression of KRAB domain-containing genes within progenitor
subsets.
Expression profiles of additional KRAB domain-containing putative transcriptional
repressors from HSC to MEP (green), GMP (red), PreB (purple), and PreT (blue).
Accompanies Results Section: Transcriptional regulation of HSCs
Supplementary Figure 4: ImmGen module analysis identifies putative regulators of
hematopoietic stem cells and endothelial cells.
(a) Heatmap representation of all the genes of ImmGen module 40 with HSC enriched
genes indicated on the left.
(b) Heatmap representation of 32 regulators for the four HSC-enriched modules.
Accompanies Results Section: G-CSF mobilization induces common transcriptional changes in
HSCs and multi-potent progenitors
Supplementary Figure 5: The transcriptome of HSCs purified by either the Slam code or
CD34/Flk2 immunophenotypic strategies largely overlap.
(a) Flow cytometry plots showing that HSCs sorted by LKS 34-Flk2- or LKS
CD150+ CD48- significantly overlap.
(b) Expression profiling plot which shows that vast majority of genes are similarly
expressed between the 2 differently sorted HSC populations.
(c) Shows that only 24 probes differ significantly between the two HSC types.
(d) Principle component analysis showing both HSC populations reside in similar
space.
Supplementary Figure 6: Mobilized HSCSlam and MPPSlam gene signatures.
(a) Pair-wise analysis showing 42 genes that differ between mobilized and nonmobilized
HSCs.
(b) Proximal promoter analysis of the 42 genes from (a) identified 3 enriched
motifs.
(c) IPA analysis of 42 genes showing enriched categories.
(d) Pair-wise analysis showing 182 genes differentially expressed genes between
mobilized and non-mobilized MPP cells.
(e) Proximal promoter analysis of the 182 genes from (d) identified 3 enriched
motifs.
(f) IPA analysis of 42 genes showing enriched categories.
Accompanies Results Section: HLF is a positive regulator of multi-lineage potential and self-renewal in
vitro
Supplementary Figure 7: Continued Hlf expression is required for continued serial plating
potential.
Withdrawal of Dox (turning OFF Hlf) results in loss of Hlf phenotype, indicating Hlf effect is not the
result of a cell transformation cell and that continued Hlf expression is required for serial replating.
Supplementary Tables
Supplementary Table 1: Hematopoietic and Progenitor Cell Nomenclature.
Correlates ImmGen cell nomenclatures utilized within the manuscript to those previously published.
Supplementary Table 2: HSPC Induced Gene List.
Supplementary Table 3: Multipotent Cell Induced Gene List.
Supplementary Table 4: HSC Induced Gene List.
Supplementary Table 5: Differentially Expressed Genes Between Steady State and Mobilized
HSPCs.
Supplementary Table 6: Differentially Expressed Genes Between Steady State HSCSLAM
and moHSCSLAM populations.
Supplementary Table 7: Differentially Expressed Genes Between Steady State MPPSLAM and
moMPPSLAM populations.
B.MZ.Sp !
T.8Nve.Sp !
T.8Eff.Sp.OT1.48hr.LisOva !
T.8Eff.Sp.OT1.24hr.LisOva !
T.8Eff.Sp.OT1.12hr.LisOva !
-1!
ncRNA metabolic!
T.8Nve.Sp.OT1 !
-0.5!
T.DPbl.Th !
0!
T.4Nve.Sp !
0.5!
T.ISP.Th !
1!
T.DPsm.Th !
cell cycle!
T.DN4.Th !
B cells!
2.5!
preT.DN3A.Th !
2!
preT.DN2.Th !
b
preT.ETP.Th !
B.Pl.AA4p220n.BM !
B.Pl.AA4p220p.BM !
1.5!
B.Fo.Sp !
ncRNA metabolic!
B.GC.Sp !
NT
B.T2.Sp!
T
B.T3.Sp !
NK
B.T1.Sp!
B
B.FrF.BM!
DC
B.FrE.BM!
Mo G
preB.FrD.BM!
MF
preB.FrC.BM!
a
proB.FrBC.BM!
HSPC
Normalized category value
Supplementary 1
T gd
c
3!
cell cycle!
2!
T cells!
1.5!
1!
0.5!
0!
-0.5!
-1!
Supplementary 2
Population
Name
HSCs
MPPs
Oligopotent Progenitors
ImmGen
LT34, LTSL,
Common
HSC, HSC-Slam, MPP-Slam, MPP1, MPP2, CMP, MEP, GMP, MDP, CDP, MLP, CLP
HSPC
MF
STSL,
Mo
G
ST34, MPP34, CMP, MEP, GMP, MDP, CDP, MLP, CLP
DC
B
NK
T
NT
T gd
Array Value (linear)
Supplementary 3
Zfp192
Zfp111
Zfp874
Zfp788
Zfp113
Zfp40
Zfp799
Gm7036
Zfp455
Zfp57
Rex2
Zfp945
Zfp286
Zfp30
Zfp820
Zfp808
HSC MPP1 MPP2 CMP MEP
HSC MPP1 MPP2 CMP GMP
HSC MPP1 MPP2 CMP MEP
HSC MPP1 MPP2 CMP GMP
HSC MPP1 MPP2 CMP MEP
HSC MPP1 MPP2 CMP GMP
HSC MPP1 MPP2 CMP MEP
HSC MPP1 MPP2 CMP GMP
HSC MPP1 MPP2 CLP PreB
HSC MPP1 MPP2 CLP PreT
HSC MPP1 MPP2 CLP PreB
HSC MPP1 MPP2 CLP PreT
HSC MPP1 MPP2 CLP PreB
HSC MPP1 MPP2 CLP PreT
HSC MPP1 MPP2 CLP PreB
HSC MPP1 MPP2 CLP PreT
Supplementary 4
Stromal
ImmGen
LT34, LTSL, STSL, LTSL . FL,STSL.FL, ST34, MPP34, CMP, MEP, GMP, MDP, CDP, MLPs, MLPs.FL CLP, CLP.FL
Common
HSC, HSC-Slam, MPP-Slam,
a
HSPC
G
MF
Mo
MPP1, MPP2, CMP, MEP, GMP, MDP, CDP,
DC
B
NK
MLP,
T
CLP
NKT
F
Fibroblast
T gd
Endothelial
F F
EC S
Gulp1
Cdc42bpa
Myct1
Scarf1
Grb10
Mycn
Fzd6
Erg
Zfp521
Ppic
Afap1l1
Hspa12b
Bc020535
Emcn
Eltd1
Nbea
Ttpa
Tek
Tie1
2610305d13Rik
Gng11
Abcg2
Fgd5
Sgce
Ica1
Cadps2
Csgalnact1
Pdgfd
Esam
Rbp1
Shroom4
Armcx1
Maged2
6230427J02Rik
9430020K01Rik
Aqp1
Btbd3
Caskin2
CD34
CD59a
Darc
Dnmt3b
Egfl7
Etl4
Fam171a1
Fam65a
Gja1
Gmpr
Gpr56
Hecw2
Hmga2
Lphn2
Magl1
Mansc1
Me1
Mprip
Mtap1b
Pcdh7
Pcp4l1
Pkn3
Plcb4
Selp
Shank3
Syde1
Thsd1
Tjp1
Tmem98
HSCinduced
genes
-3
b
S
0
HSPC
G
MF
+3
Mo
DC
B
NK
T
NKT
T gd
F F
EC S
Hlf
Erg
Mycn
Sox4
Stat1
Tfdp2
Sp100
Ruvbl2
Gabpb1
Smad5
Gata2
Smyd3
Nfe2
Lmo4
Morf4l2
Plag1
Cbx7
Nab1
Tcfap4
Prdm5
Hdac6
Mecom
Hoxa9
Thra
Runx3
Nr2c1
Chd7
Cby1
Bcl3
Rcor2
E2f1
Hnf4a
Gata1
Insm1
Pou2f2
Rara
Gata4
Wwtr1
Cebpe
Klf15
Tbx21
Sox7
Gata6
Supplementary 5
Mt1
Myc
Leprot
Rpp38
Lyar
Nampt
Pgrmc2
Car1
Ap3S1
Loc641050
Igj
Gpr171
Qser1
Ctsh
Dennd4a
Capza1
Ndn12
Snora21
2400001E08Rik
Slc25a36
Mylpf
Gm6375
Gm3148
Zfp407
-3
0
+3
Supplementary 6
!"#$%$&'()*+,+%-.)/'0')1$/0-234'5!!
Egr1, Sp1, Pax4, Zfp281, Zfp740, Gabpa, Ascl2
-3
0
+3
d
e
Sna, Rpn4, Ctcf, Tcfe2a, Hap4, Esrra
Rim101
Zfp105, Azf1, Afl1, Elf3, Mtf1
-3
f
0
+3
Colony number /10k plated cells
Supplementary 7
*""!
)""!
+,-..!
Quaternary plating
+-/+.0!
(""!
+,/+./+,.!
'""!
&""!
%""!
$""!
#""!
"!
DOX:
-
+
HSC + HLF
-
+
MPPs+ HLF
-
+
MyPro + HLF
Supplementary Figure Legends
Supplementary Fig. 1. Enriched genes within hematopoietic stem and progenitor
cells. Heatmap representation of all 1605 significantly enriched genes in HSPCs across
ImmGen datasets (a) with red indicating high expression, and blue low expression. Cell
types broadly grouped together are indicated and color-coded. Enlarged key indicates
the HSPC cell types by the population ImmGen names, and the names used in this
study. Graphs for B cell (b) and T cell (c) showing the averages-categories nc-RNA
metabolic (Blue) and Cell Cycle (Red) for the indicated cell types.
Supplementary Fig. 2. Enriched genes within multipotent stem and progenitor
cells. Heatmap representation of all 443 significantly enriched genes in multi-potent
progenitors across ImmGen datasets with red indicating high expression, and blue low
expression. Cell types broadly grouped together are indicated and color-coded. Enlarged
key indicates the HSPC cell types by the population ImmGen names, and the names
used in this study
Supplementary Fig. 3. Expression of KRAB domain-containing genes within
progenitor subsets. Graphs showing the linear-values (averaged arrays replicates +/SEM) of the indicated genes along differentiation trajectories from HSC to MEP (green),
GMP (red), PreB (purple), and PreT (blue). Biological replicates: n=2 (MPP1, MPP2,
MEP), n=3 (HSC, CMP, GMP, PreB, PreT), n=4 (CLP).
Supplementary Fig. 4. ImmGen module analysis identifies putative regulators of
hematopoietic stem cells and endothelial cells. (a) Heatmap representation of all the
genes of ImmGen module 40 with the HSC-enriched genes indicated on the left. Keys
indicate the cell types that include stromal cells in addition to hematopoietic cells. (b)
Expression of all of the predicted regulators in each of the modules enriched for HSC-
enriched genes. The Log2 values are shown on a scale from lowest (black) to highest
(violet).
Supplementary Fig. 5. The transcriptome of HSCs purified by either the Slam code
or CD34/Flk2 immunophenotypic strategies largely overlap. (a). Multi-color FACS
demonstrating that HSCs immunophenotypically defined by KLSCD48-CD150+ largely
overlap with HSCs defined by LSKFlk2-CD34-. b). Mean class expression analysis of
HSCs purified by the two strategies described in (a) showing that they are
transcriptionally very similar (Hochberg test; FDR<10%, and fold-change >2) (c).
Heatmap representation of the differentially expressed genes between HSCs purified by
the two strategies described in (a) (FDR<10% and fold-change >2). (red: up-regulated
and blue: down-regulated genes in the indicated data set). (d). Population-distance
analysis of microarray data is presented within 3 principal-components (PC 1, 2 and 3).
Each point represents a single array. Related cell types are color-coded.
Supplementary Fig. 6. Mobilized HSCSlam and MPPSlam gene signatures. (a).
Heatmap representation of differentially expressed genes (91 genes) in mobilized
HSCSlam vs. nonmobilized HSCSlam. (FDR<10%) (red: up-regulated and blue: downregulated genes in the indicated data set). (b). Statistically over-represented
transcription factor binding motifs (TFBs) in promoter regions (+1K to -1K of the TSS) of
differentially expressed genes in mobilized HSCSlam. On the top of each motif,
information is given about the putative TF family binding motif and the P-value before the
null model correction. (c). Biological functions for genes differentially expressed in
mobilized HSCSlam vs. nonmobilized HSCSlam identified by using Ingenuity Pathway
Analysis (IPA) (p value <0.05 as calculated by Fisher’s test). (d). Heatmap
representation of differentially expressed genes (502 genes) in mobilized MPPSlam vs.
nonmobilized MPPSlam. (FDR<10%) (red: up-regulated and blue: down-regulated genes
in the indicated data set). (e). Statistically over-represented transcription factor binding
motifs (TFBs) in promoter regions (+1K to -1K of the TSS) of differentially expressed
genes in mobilized MPPSlam. On the top of each motif, information is given about the
putative TF family binding motif and the P-value before the null model correction. (f).
Biological functions for genes differentially expressed in mobilized MPPSlam vs.
nonmobilized MPPSlam identified by using Ingenuity Pathway Analysis (IPA) (p value
<0.05 as calculated by Fisher’s test).
Supplementary Fig. 7. Continued HLF expression is required for continued serial
plating potential. Results of quaternary plating of HLF-transduced stem and progenitor
cells plated in methylcellulose in the presence (+) or absence (-) of doxycycline (-Dox).
Colony numbers and colony types are indicated. Three biological replicates per sample,
and error bars indicate SEM.
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