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.
a HSPC B DC MF Mo G NK T NT T gd
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B.T2
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B.Pl
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p220
p.BM
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Pl.A
A4p2
20n.
BM !
B cells!
ncRNA metabolic! cell cycle!
-1!
-0.5!
0!
0.5!
1!
1.5!
2!
2.5!
3!
preT
.ETP
.Th !
preT
.DN2
.Th !
preT
.DN3
A.Th
!T.
DN4.
Th !
T.IS
P.Th
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DPsm
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T.DP
bl.T
h !
T.4N
ve.S
p !
T.8N
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T cells!
ncRNA metabolic! cell cycle!
Supplementary 1
b c
Nor
mal
ized
cat
egor
y va
lue
HSPC B DC MF Mo G NK T NT T gd
Supplementary 2
LT34, LTSL, STSL, ST34, MPP34, CMP, MEP, GMP, MDP, CDP, MLP, CLP
HSCs MPPs Oligopotent Progenitors
HSC, HSC-Slam, MPP-Slam, MPP1, MPP2, CMP, MEP, GMP, MDP, CDP, MLP, CLP
Population Name
ImmGen
Common
Supplementary 3
HSC HSC HSC HSC
MPP1 MPP1 MPP1 MPP1
MPP2 MPP2 MPP2 MPP2
MEP GMP PreB PreT
CMP CMP CLP CLP
HSC HSC HSC HSC
MPP1 MPP1 MPP1 MPP1
MPP2 MPP2 MPP2 MPP2
MEP GMP PreB PreT
CMP CMP CLP CLP
HSC HSC HSC HSC
MPP1 MPP1 MPP1 MPP1
MPP2 MPP2 MPP2 MPP2
MEP GMP PreB PreT
CMP CMP CLP CLP
HSC HSC HSC HSC
MPP1 MPP1 MPP1 MPP1
MPP2 MPP2 MPP2 MPP2
MEP GMP PreB PreT
CMP CMP CLP CLP
Arr
ay V
alue
(lin
ear)
Zfp455
Zfp286
Zfp57
Zfp30
Rex2
Zfp820
Zfp945
Zfp808
Zfp113 Zfp40
Zfp111 Zfp192 Zfp874
Zfp799 Gm7036
Zfp788
S F HSPC B DC MF Mo G NK T NKT T gd F EC
S F HSPC B DC MF Mo G NK T NKT T gd F EC
Stromal
Fibroblast Endothelial S F
a
b
Supplementary 4
-3 +3 0
LT34, LTSL, STSL, LTSL . FL,STSL.FL, ST34, MPP34, CMP, MEP, GMP, MDP, CDP, MLPs, MLPs.FL CLP, CLP.FL HSC, HSC-Slam, MPP-Slam, MPP1, MPP2, CMP, MEP, GMP, MDP, CDP, MLP, CLP
ImmGen
Common
HSC- induced genes
Gulp1 Cdc42bpa Myct1 Scarf1 Grb10 Mycn Fzd6 Erg Zfp521 Ppic Afap1l1
Sgce
Hspa12b Bc020535 Emcn Eltd1 Nbea Ttpa Tek Tie1 2610305d13Rik Gng11 Abcg2 Fgd5
Ica1 Cadps2 Csgalnact1
Gmpr
Pdgfd Esam Rbp1 Shroom4 Armcx1 Maged2 6230427J02Rik 9430020K01Rik Aqp1 Btbd3 Caskin2 CD34 CD59a Darc Dnmt3b Egfl7 Etl4 Fam171a1 Fam65a Gja1
Gpr56 Hecw2 Hmga2 Lphn2 Magl1 Mansc1 Me1 Mprip Mtap1b Pcdh7 Pcp4l1 Pkn3 Plcb4 Selp Shank3 Syde1 Thsd1 Tjp1 Tmem98
Lmo4
Hlf Erg Mycn Sox4 Stat1 Tfdp2 Sp100 Ruvbl2 Gabpb1 Smad5 Gata2 Smyd3 Nfe2
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
+3 -3 0
Mt1 Myc Leprot Rpp38 Lyar Nampt Pgrmc2 Car1 Ap3S1 Loc641050 Igj Gpr171 Qser1 Ctsh Dennd4a Capza1 Ndn12 Snora21 2400001E08Rik Slc25a36 Mylpf Gm6375 Gm3148 Zfp407
!"#$%$&'()*+,+%-.)/'0')1$/0-234'5!!Supplementary 6
d e
f +3 -3 0
+3 -3 0
Egr1, Sp1, Pax4, Zfp281, Zfp740, Gabpa, Ascl2
Sna, Rpn4, Ctcf, Tcfe2a, Hap4, Esrra
Rim101
Zfp105, Azf1, Afl1, Elf3, Mtf1
Col
ony
num
ber /
10k
plat
ed c
ells
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Quaternary plating
HSC + HLF
Supplementary 7
MPPs+ HLF MyPro + HLF DOX: - + - + - +
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: down-
regulated 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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