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Cell Stem Cell, Volume 3

Supplemental Data

Hematopoietic Stem Cell Quiescence

Is Maintained by Compound Contributions

of the Retinoblastoma Gene Family

Patrick Viatour, Tim C. Somervaille, Shivkumar Venkatasubrahmanyam,

Scott Kogan, Margaret E. McLaughlin, Irving L. Weissman, Atul J. Butte,

Emmanuelle Passegué, and Julien Sage

SUPPLEMENTAL DATASET

Hematopoietic stem cell quiescence is maintained by compound contributions of the

retinoblastoma gene family – Viatour et al.

Figure S1 page 2 Legend Figure S1 page 3 Figure S2 and legend page 4 Figure S3 page 5 Legend Figure S3 page 6 Figure S4 page 7 Legend Figure S4 page 8 Figure S5 page 9 Legend Figure S5 page 10 Figure S6 page 11 Legend Figure S6 page 12 Supplemental Methods (microarrays) page 13

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Figure S1: Rb deficient mice display extramedullary hematopoiesis and a myeloproliferation.

Rosa26-CreERT2 Rblox/lox mice were analyzed 6 weeks after tamoxifen injection (see main text

for references to Rosa26-CreERT2 mice). Controls were Rblox/lox mice injected with the same

dose of tamoxifen. A Splenic cellularity of control (CT) and induced Rosa26-CreERT2 Rblox/lox

mice (Rb KO) - n=3. B Representative cytospins from the bone marrow (BM) and the spleen

of control (CT, left) and Rosa26-CreERT2 Rblox/lox mice (Rb KO, right) show an increase in the

number of cells from the neutrophil lineage in the BM and an extramedullary hematopoiesis in

the spleen of mutant mice. C Representative immunophenotypic analysis of KLS cells,

myeloid progenitors (MP), lymphoid progenitors (CLP) and mature myeloid cells by FACS in

the bone marrow (BM, upper rows) and the spleen (lower rows) of control and Rb mutant

mice. In the mature myeloid cells compartment, Rb mutant mice exhibit an increase in the

Mac-1+, Gr-1+ population. D Cell counts of hematopoietic subpopulations in the BM (upper

panels) and in the spleen (lower panels) of control (CT, white) and Rb mutant mice (Rb KO,

grey) mice (n=3). Progenitors in left panels: KLS: Lineageneg, c-Kit+, Sca1-; MP: Myeloid

Progenitors; CMP: Common Myeloid Progenitors; GMP: Granulocytes-Monocytes

Progenitors; MEP: Megakaryocyte-Erythrocytes Progenitors; CLP: Common Lymphoid

Progenitors. Mature populations in right panels: pre-M: Mac-1+, Gr-1low/interm cells; Gr:

Granulocytes, Mac-1+, Gr-1+; B: B cells; E: Erythrocytes. Note the increase in granulocytes in

the BM, indicative of myeloproliferation and the increase in the number of hematopoietic

progenitors in the spleen, indicative of extramedullary hematopoiesis. E Methylcellulose

assays with 10,000 unfractioned BM cells or 50,000 unfractioned splenocytes from control

(CT) and Rb mutant (Rb KO) mice. Colonies were counted 8-10 days after plating and the

numbers represent the average of 3 independent experiments performed in duplicate. No

significant difference was observed in the number of colonies between control and mutant BM

cells (left). The increase number of colonies growing from Rb mutant splenocytes is indicative

of an extramedullary hematopoiesis. Data are mean ± SD.

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Figure S2: Development of a myeloid disease in Rb family TKO mice.

A QPCR analysis of Rb, p130, and p107 mRNA levels in the bone marrow (BM) of control

(CT, white) and Mx1-Cre TKO mice (TKO, black) two months after pI-pC treatment (n=3). B

Cytospins on sorted pre-M (Mac-1+/Gr-1interm) and mature granulocytes (Gr, Mac-1+/Gr-1+)

(right). C FACS analysis of erythrocytes (E, c-Kit/Ter119; left) and B cells (B, B220/CD19;

right) in the BM of representative mice. D Analysis of myeloid (M, left), erythroid (E, middle)

and B cell (B, right) populations in the spleen of representative control and mutant mice. Data

are mean ± SD.

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Figure S3: Extramedullary erythropoiesis in Rb family TKO mice.

A Erythrocytes from control (left) and Mx1-Cre TKO (right) BM were fractioned into three

subpopulations based on FSC, SSC and CD45 expression (left panels). These three

subpopulations correspond to different stages in the maturation of the erythrocyte lineage (A-

early, B-intermediate, C-late differentiation stages). Each subpopulation was then analyzed

for Ter119 and CD71 expression (right panels), two additional markers of erythrocyte

differentiation. B BM cellularity for the three subpopulations (A, B, C) (Four limbs, n=5 mice).

C Same as A, in the spleen. D Average spleen cellularity (n=2) for the three splenic

subpopulations. Data are mean ± SD.

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Figure S4: Short term consequences of loss of the Rb gene family for hematopoiesis.

A Representative FACS analysis of progenitor populations in the bone marrow (BM) of control

(CT) and Rosa26-CreERT2 TKO mice (TKO) two weeks after tamoxifen injection: KLS

(Lineageneg, c-Kit+, Sca1+ – MPP: CD34+, Flk2+ – ST-HSC: CD34+, Flk2- – LT-HSC: CD34-,

Flk2-), Myeloid Progenitors (MP: Lineageneg, c-Kit+, Sca1- – GMP: FcgRhigh, CD34+ – CMP:

FcgR+, CD34+ – MEP: FcgRlow, CD34low) and CLP (Lineageneg, Flk2+, IL7Ra+). B

Representative analysis of myeloid (Mac-1/Gr-1) and B cells (B220) mature lineages in the

BM of control and Rosa26-CreERT2 TKO mice. Note the expansion of pre-M cells (21%

versus 7.7%). In C and D, recipient mice (CT and TKO, Ly5.1+) were transplanted with

unfractioned BM from wild-type mice (WT, Ly5.1+/Ly5.2+). The analysis was performed two

weeks after tamoxifen treatment (n=4). C Representative analysis of KLS and MP progenitor

populations. D Myeloid cells were analyzed with Mac-1/Gr-1 staining. There were no

statistical differences in the WT populations analyzed when WT cells were transplanted into

CT mice compared to TKO mice.

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Figure S5: The TKO myeloproliferation is cell autonomous.

A Transplantation strategy: Rag2 mutant mice were lethally irradiated and subsequently

transplanted with 2x106 bone marrow cells from either control condTKO or Rosa26-CreERT2

condTKO mice. After reconstitution, recipient mice were injected intraperitoneally with

tamoxifen to induce Cre activity. Mice were then aged for four months before analysis, at a

time where they were outwardly sick. B-H Histopathological analysis of the recipient mice.

Skin (B, C – 100x), Liver (D, E – 200x), Spleen (F, G – 100x) from recipient mice transplanted

with either control (CT) (B, D, F) or Rosa26-CreERT2 condTKO cells (TKO) (C, E, G). TKO

recipient mice exhibited skin rash (hyperkeratosis and infiltration of blood cells, arrowhead),

their liver was infiltrated with myeloid cells (arrows), and the architecture of their spleen was

severely disrupted. H The spleen of mice with TKO cells was also enlarged, suggestive of

extramedullary hematopoiesis. The number of bone marrow cells was not significantly altered

in mutant and control animals (data not shown) I, J Mature blood populations in the bone

marrow were analyzed by flow cytometry (n=3). The number of pre-M cells was significantly

increased in TKO recipients versus controls, and B cell populations were strongly decreased,

further indicating that the recipient mice with TKO cells develop a myeloproliferation similar to

TKO mice and that this disease is intrinsic to the mutant cells. Data are mean ± SD.

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Figure S6: Expression levels of self-renewal genes in TKO and control KLS cells.

A Genes associated with self-renewal ability were not significantly decreased in TKO KLS

cells (data from the microarrays characterized in Figure 7). The heat map shows the

expression of genes with cell cycle and self-renewal-related functional annotations identified

through a literature search. The rows correspond to genes and the columns to samples.

Gene expression values are indicated on a color scale according to the color scheme shown.

B Expression levels of four of the genes present in the microarrays were further quantified by

QPCR on independent RNA samples. There were no significant differences between control

(CT) and TKO samples. Data are mean ± SD.

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Supplemental Experimental Procedures

Microarray analysis

A total of 10,000 KLS cells from induced Mx1-Cre TKO mice were sorted into RNALater

(Ambion). Controls were obtained from Rblox/lox;p130lox/lox;p107+/- mice; each control sample

represents a pool of cells obtained from two control mice. RNA was isolated with Trizol and

twice amplified with a RiboAmp RNA amplification kit (Arcturus Engineering, Mountain View,

CA). Amplified cRNA was streptavidin-labeled, fragmented, and hybridized to Affymetrix 430-

2.0 arrays as recommended by the manufacturer (Affymetrix, Santa Clara). Arrays were

scanned with a Gene Chip Scanner 3000 (Affymetrix) running GCOS 1.1.1 software

Quantitative PCR experiments. Amplification and subsequent processing were performed at

the Stanford Microarray facility. Raw data will be available for download from Gene

Expression Omnibus (http://ncbi.nlm.nih.gov/geo, Accession Number GSE11253).

Data from 3 biological replicates of each genotype was analyzed using R (cran.r-

project.org) and Bioconductor (www.bioconductor.org) software. Background correction,

normalization, and calculation of gene expression values by model-fitting were performed

using the Robust Multiarray Average procedure (Irizarry et al., 2003). Hierarchical clustering

of the 6 samples based on pairwise correlation across all probe sets indicated that one of the

3 control samples was an outlier (high background and anomalous control probe sets); this

sample was excluded from further analysis. Genes which were differentially expressed in

TKO and control cells were identified using Significance Analysis of Microarrays (Tusher et

al., 2001) with the false-discovery rate limited to 10%. These gene lists were examined for

the enrichment of functional categories using DAVID (http://david.abcc.ncifcrf.gov/).

Supplemental References Irizarry, R. A., Hobbs, B., Collin, F., Beazer-Barclay, Y. D., Antonellis, K. J., Scherf, U., and

Speed, T. P. (2003). Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4, 249-264.

Tusher, V. G., Tibshirani, R., and Chu, G. (2001). Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A 98, 5116-5121.