Rb Regulates Interactions between Hematopoietic Stem Cells and Their Bone Marrow Microenvironment

Rb Regulates Interactions betweenHematopoietic Stem Cells and TheirBone Marrow MicroenvironmentCarl R. Walkley,1 Jeremy M. Shea,1 Natalie A. Sims,2 Louise E. Purton,3 and Stuart H. Orkin1,4,*1Department of Pediatric Oncology, Dana-Farber Cancer Institute, Division of Hematology/Oncology and Stem Cell Program,

Children’s Hospital Boston, Harvard Stem Cell Institute, Harvard Medical School, Boston, MA 02115, USA2St. Vincent’s Institute of Medical Research and Department of Medicine at St. Vincent’s Hospital, The University of Melbourne,

Fitzroy, Victoria 3065, Australia3Center for Regenerative Medicine, Massachusetts General Hospital, Harvard Stem Cell Institute, Boston, MA 02114, USA4Howard Hughes Medical Institute, Boston, MA 02115, USA

*Correspondence: [email protected] 10.1016/j.cell.2007.03.055

SUMMARY

Hematopoiesis is maintained by stem cells(HSCs) that undergo fate decisions by integrat-ing intrinsic and extrinsic signals, with the latterderived from the bone marrow (BM) microenvi-ronment. Cell-cycle regulation can modulatestem cell fate, but it is unknown whether thisrepresents an intrinsic or extrinsic effector offate decisions. We have investigated the roleof the retinoblastoma protein (RB), a centralregulator of the cell cycle, in hematopoiesis.Widespread inactivation of RB in the murinehematopoietic system resulted in profoundmyeloproliferation. HSCs were lost from theBM due to mobilization to extramedullary sitesand differentiation. This phenotype was notintrinsic to HSCs, but, rather, was the con-sequence of an RB-dependent interactionbetween myeloid-derived cells and the micro-environment. These findings demonstrate thatmyeloproliferation may result from perturbedinteractions between hematopoietic cells andthe niche. Therefore, RB extrinsically regulatesHSCs by maintaining the capacity of the BMto support normal hematopoiesis and HSCs.

INTRODUCTION

Under homeostatic conditions, the adult hematopoietic

system is maintained by a small number of stem cells

(HSCs) that reside in the bone marrow in a specialized

microenvironment, termed the niche (Adams and Scad-

den, 2006; Schofield, 1978). It is within the niche that

HSCs undertake fate decisions, including differentiative

divisions to generate progenitor cells and self-renewal

divisions necessary to sustain HSCs throughout life.

Both intrinsic and extrinsic cues are integrated within the

niche to maintain effective control over HSCs, ensuring

contribution to hematopoiesis without aberrant prolifera-

tion (Fuchs et al., 2004; Moore and Lemischka, 2006).

Whereas the majority of HSCs are in a slowly dividing

state, termed relative quiescence, with a cell-division

cycle in the mouse in the range of 2–4 weeks, progenitor

cells exhibit rapid cycling (Bradford et al., 1997; Passegue

et al., 2005). HSCs can also be stimulated to rapidly enter

the cell cycle and contribute to hematopoiesis (Li and

Johnson, 1994). In part, the dramatic contrast in cell-cycle

status between stem and progenitor cells has led to the

hypothesis that cell-cycle regulation plays a fundamentally

important role in stem cell fate determination.

Decisions to enter the cell cycle are regulated by the

G1-S phase restriction point (Sherr and Roberts, 2004).

The sequential phosphorylation and subsequent inactiva-

tion of the retinoblastoma protein (RB) is an important part

of this transition (Weinberg, 1995). RB is phosphorylated

by cyclin-cyclin-dependent kinase (Cdk) complexes. Sev-

eral negative regulators of Cdk activity have been studied

in the context of HSC biology. Loss of the Cdk2 inhibitors

p21Cip1 and p27Kip1 revealed a divergent role in HSC reg-

ulation, with loss of p21Cip1 resulting in a subtle increase in

sensitivity to stress-induced exhaustion apparent in vivo

after quaternary transplant (Cheng et al., 2000). Loss of

p27Kip1 resulted in a 2-fold increase in the number of

long-term repopulating HSCs in addition to an enlarged

progenitor compartment (Walkley et al., 2005). Loss of

both Cdk4/6 inhibitors p16Ink4a and p19ARF revealed

a small increase in serial transplant potential (Stepanova

and Sorrentino, 2005), with a similar phenotype observed

in p16Ink4a single mutant HSCs (Janzen et al., 2006). Loss

of p18Ink4c resulted in increased HSC repopulation and

frequency (Yuan et al., 2004).

Collectively, these studies suggest that negative cell-

cycle regulators that impact directly on RB-family protein

function may influence HSC fate. It is indeterminate if

Cell 129, 1081–1095, June 15, 2007 ª2007 Elsevier Inc. 1081

these phenotypes reflect intrinsic or extrinsic effects on

HSCs and hematopoiesis, as all studies to date have

utilized nonconditional mutant alleles that are not hemato-

poietic restricted in their effects. The analysis of HSCs

from germ-line deficient animals does not allow for the

clear delineation of intrinsic and extrinsic contribution to

the observed HSC phenotype. Such studies have largely

not accounted for effects on HSC genesis or potentially

defective niche support that affect HSCs prior to trans-

plantation analysis. While serial transplant studies are

suggestive of an intrinsic role for Cdkis in HSC biology,

they do not exclude a role for the environment from which

these cells were removed, necessitating analysis utilizing

hematopoietic restricted deletion. Indeed, a recent study

demonstrated that the p27Kip1�/� microenvironment me-

diates lymphoid expansion observed in the p27Kip1�/�

animals, possibly indicating that the HSC expansion

observed in p27Kip1�/� bone marrow is extrinsic in nature

(Chien et al., 2006; Walkley et al., 2005). This result sug-

gests that cell-cycle regulators may play a role in regulat-

ing the competence of the hematopoietic niche in addition

to intrinsic roles in HSC fate determination.

Recent studies have begun to characterize the adult

bone marrow niche (Schofield, 1978). Osteoblasts appear

to comprise an important component of the HSC niche, as

modulation of osteoblast number and function influences

hematopoiesis and HSC fate via extrinsic mechanisms

(Calvi et al., 2003; Visnjic et al., 2004; Zhang et al., 2003).

Additionally, numerous extrinsic factors modulate HSC

function. These factors include retinoic acid, extracellular

calcium, osteopontin, angiopoietins, and Notch ligands

(Adams et al., 2006; Arai et al., 2004; Purton et al., 2000;

Stier et al., 2005; Varnum-Finney et al., 1998; Zhang

et al., 2006a). Extrinsic regulation of homeostatic HSC

numbers can be dominant to intrinsic cues in vivo. For

example, HSCs engineered to overexpress HoxB4 expand

in vivo only to the level of normal HSCs despite markedly

enhanced in vitro self-renewal and proliferative capacity

(Krosl et al., 2003). Additionally, systemic factors con-

tained in the peripheral blood of young animals may

reactivate self-renewal-associated pathways in progeni-

tors of older animals, suggesting an important role for

extrinsic signaling in stem cell regulation (Conboy et al.,

2005). While these studies have begun to define the

bone marrow niche, little is currently known regarding

molecular regulators of the niche and their role in influenc-

ing HSC fate decisions. Regulatory interactions between

the hematopoietic cells and the nonhematopoietic-derived

microenvironment are largely unknown. Moreover, the

regulators of these potential interactions and how they

affect hematopoiesis and HSC function are unexplored.

Here we have utilized a conditional deletion strategy to

investigate the role of the RB in the regulation of adult HSC

fate. We found that widespread inactivation of Rb resulted

in the development of a myeloproliferative disease, char-

acterized by extramedullary hematopoiesis and mobiliza-

tion of primitive cells into the periphery. HSCs were lost

from the BM as a result of increased differentiation and

1082 Cell 129, 1081–1095, June 15, 2007 ª2007 Elsevier Inc.

mobilization from the BM. The phenotype is not HSC

intrinsic, as it was not recapitulated upon inactivation of

RB in HSCs maintained in a wild-type environment (Walk-

ley and Orkin, 2006). Strikingly, however, concomitant

deletion of Rb from myeloid-derived cells and the micro-

environment generated the myeloproliferative disorder,

thereby demonstrating that RB is an essential regulator

of the interaction between myeloid-derived cells and the

BM microenvironment. Thus, RB extrinsically controls

HSCs by maintaining the competence of the BM to sup-

port normal HSCs and hematopoiesis.

RESULTS

Rb Deletion Leads to Myleoproliferation

Rb was inactivated in hematopoietic cells, including

HSCs, using the interferon-inducible Mx-Cre transgene

and pRbfl/fl animals (Kuhn et al., 1995; Sage et al., 2003;

Walkley and Orkin, 2006). We performed PCR on both

genomic DNA and cDNA from whole BM samples to

confirm Rb deletion (BM, Figures 1A and 1B). Rb was

quantitatively and stably deleted from hematopoietic

cells, and expression of the related p130 and p107 was

not altered as a result of Rb loss. Thus, with this condi-

tional system, we achieve specific loss of Rb without

compensatory gain of expression of other genes coding

for pocket proteins.

Analysis of the peripheral blood of control (Mx-Cre�pRbfl/fl,

pIpC injected) and RbD/D animals following pIpC treatment

revealed that RbD/D animals developed a mild but stable

anemia immediately following Rb deletion (C.R.W and

S.H.O, unpublished data) and by 6 weeks developed throm-

bocytosis (Figure 1C). By 4 weeks, RbD/D animals developed

pan-leukocytosis (Figure 1D) that was accompanied by

elevated levels of circulating progenitor cells, as determined

by in vitro colony-forming capacity (CFU-GEMM and CFU-

G/GM) and phenotypic staining (lin�c-Kit+Sca-1+, LKS+;

Figures 1E and 1F; Okada et al., 1992). Although leukocytosis

was apparent by 4 weeks post-Rb deletion, increased circu-

lating progenitors could be detected as early as 2 weeks after

pIpC (LKS+ increased 3.7-fold, p % 0.01, n = 7 per genotype;

CFU-GEMM increased 2.4-fold, p % 0.01, CFU-M/GM

increased3.9-fold,p%0.01,n=6pergenotype).Surprisingly,

the levels of circulating progenitors were comparable to those

achieved during pharmacologically induced mobilization of

stem and progenitors in the C57Bl/6 strain background

(Ghiaur et al., 2006). However this was a chronic, rather than

an acute, response in the RbD/D mutant.

BM cellularity was not initially altered; however, at 12

weeks post-pIpC it was increased by 40% in RbD/D

animals (Figure 2A). We observed the rapid development

of a myeloproliferative-like disease within the bone

marrow. The phenotype was fully penetrant and was char-

acterized by myeloid hyperplasia (predominantly neutro-

philia) and suppression of both B-lymphopoiesis and

erythropoiesis (Figures 2B, S1, and S2). Phenotypic

stem and primitive progenitor populations (LKS� and

LKS+) were increased significantly in the BM of RbD/D

Figure 1. Rapid Mobilization of Primitive Cells into the Peripheral Blood Following Deletion of Rb

(A) Genomic PCR on whole BM from control (Mx�pRbfl/fl) and Rb-deficient animals (Mx+pRbD/D) at 6 and 12 weeks post-pIpC.

(B) qRT-PCR for pRb, p130, and p107 on cDNA of control and Rb-deficient animals (n = 3 independent samples) 12 weeks post-pIpC.

(C) Platelets and (D) leukocytes in PB following Rb deletion (time 0 = final dose of pIpC); n R 4/time point; *p < 0.05.

(E) Day 12 CFU-GEMM and CFU-GM/M from the PB of at 12 weeks post-pIpC; n R 9/genotype; *p < 0.01. Value inside bars represents fold increase.

(F) FACS profile and mean number of Lin�c-Kit+Sca-1+ (LKS+) in the PB; n > 4/genotype; *p < 0.01. Methylcellulose plates from day 12 of culture. Data

expressed as mean ± SEM.

animals (Figure 2C); however, the number of pheno-

typic HSCs per femur was not significantly altered

(LKS+CD34�/lo; Osawa et al., 1996; Yang et al., 2005). In

addition, RbD/D animals exhibited striking changes in the

architecture of the bone, evidenced by loss of trabecular

bone (Figure 2D). Trabecular bone is thought to represent

an important niche for HSCs within the BM (Calvi et al.,

2003). Quantitative histomorphometric analysis of the

bone at 2 weeks post-pIpC, a time point correlating with

the presence of progenitors in the peripheral blood and

spleen, demonstrated a significant reduction in trabecular

volume as a proportion of total marrow volume, a �40%

reduction in the number of trabeculae, and a doubling of

the separation of trabeculae (Figures 2E–2H).

In parallel with BM myeloproiferation, RbD/D animals de-

veloped extensive extramedullary hematopoiesis. Spleen

weight increased rapidly by 5.5-fold relative to controls

due to expanded numbers of myeloid cells, megakaryo-

cytes, and erythroid cells (Figures 2I and 2J). B- and

T cell lymphopoiesis were present at comparable levels

in RbD/D and control spleens. Phenotypic stem and pro-

genitor populations (LKS+ and LKS�) increased progres-

sively in the spleens of RbD/D animals, and by 12 weeks

were increased 45- to 50-fold (Figures 2K, S3, and S4).

Splenic architecture was effaced as a result of myeloid

and erythroid elements (Figure 2J). Hematopoietic foci

were also observed in the liver but not in the kidney

(data not shown). Despite chronic myeloproliferation, no

hematopoietic tumors have developed during the lifespan

of RbD/D mutant animals. RbD/D animals survive for ap-

proximately 8 months post-pIpC; heterozygous animals

are normal (Figures S5 and S6). Eight-month-old RbD/D

animals present with a phenotype reminiscent of hemato-

poietic failure, characterized by a significant reduction in

Cell 129, 1081–1095, June 15, 2007 ª2007 Elsevier Inc. 1083

Figure 2. Myeloproliferation Following Rb Deletion

(A) Femoral cellularity, n R 3/genotype/time point.

(B) Number of cells of each lineage/femur at 12 weeks post-pIpC; Granulocytes CD11b+Gr-1+, Macrophages CD11b+F4/80+, Immature B lymphoid

IgM�B220+, Mature B lymphoid IgM+B220+, Mature Erythroid CD71�Ter119+, and Immature Erythroid CD71+Ter119+; n R 6/genotype; *p < 0.01.

(C) Number of phenotypic HSCs (LKS+CD34�/lo) and primitive progenitors/femur; 12 weeks post-pIpC; n R 5/genotype; *p < 0.05.

(D) Representative sections of tibiae at 12 weeks post-pIpC.

(E) Volume of marrow space occupied by bone (BV/TV); 2 weeks post-pIpC; n R 13/genotype; *p < 0.05.

(F) Trabecular number/mm; *p < 0.05.

(G) Separation of trabeculae; *p < 0.05.

(H) Representative longitudinal sections of tibiae stained with Von Kossa technique (mineralized bone stained black).

(I) Spleen cellularity; n R 3/genotype/time point; *p < 0.01.

1084 Cell 129, 1081–1095, June 15, 2007 ª2007 Elsevier Inc.

spleen weight and replacement of BM by granulocytes;

however, pituitary tumors are also observed (Figure S5

and data not shown).

HSCs Are Lost from BM following Rb Deletion

RB and other negative cell-cycle regulators have been

postulated to play an important role in the regulation of

HSCs and in the subsequent hematopoiesis. However,

neither the myeloproliferative disease nor defective HSC

function was observed when Rb was deleted from HSCs

in the context of a wild-type microenvironment (Walkley

and Orkin, 2006). Given the striking phenotype we

observed when Rb was deleted from both hematopoietic

cells and the BM microenvironment, as occurs with

Mx-Cre (Zhang et al., 2003), we sought to determine the

consequences of RB loss on HSCs in these animals.

Within the BM we observed a significant increase in the

frequency of mature day 7 colony-forming cells but a de-

crease in the frequency of the more primitive in vivo day 12

colony-forming unit-spleen (CFU-S12, Figures 3A and 3B).

The numbers of both in vitro colony-forming cells (68-fold

increase in CFU-GEMM) and CFU-S8 in the spleen were

markedly increased (Figures 3C and 3D). As we observed

high levels of circulating progenitors and substantial levels

of progenitor activity in the spleen, we sought to determine

if latent HSC activity was also present in extramedullary

sites. Whole spleen cells from RbD/D animals (either 1 3

106 or 2 3 106) were transplanted with competitor whole

BM (2 3 105) into congenic recipients. At 17 weeks post-

transplant, significant multilineage repopulating activity

derived from the spleens of RbD/D animals was present,

demonstrating that functional HSCs were present in the

periphery of RbD/D mice (Figures 3E and 3F).

To determine the HSC content of the BM, we performed

limit-dilution competitive repopulation analysis with whole

BM from donor animals treated 12 weeks earlier with pIpC

(Purton et al., 2006; Szilvassy et al., 1990; Walkley et al.,

2005). Whole BM from control (Rbfl/fl) or Rb-deficient

(RbD/D, both CD45.2+) mice was mixed at varying doses

with a fixed number of competitor BM cells (CD45.1+/

CD45.2+) and transplanted into congenic recipient ani-

mals (CD45.1+). The frequency of long-term repopulating

HSCs in the RbD/D BM at 6 months posttransplant was re-

duced by 8-fold (p = 0.0005). When normalized to reflect

the increased cellularity of the RbD/D BM, this represents

a 5-fold decrease in the absolute number of HSCs per

femur (Figure 4A). Secondary transplantation demon-

strated that RbD/D HSCs were serially transplantable and

capable of stable multilineage contribution for at least

3 months. Importantly, we did not observe a progressive

decline in contribution from RbD/D HSCs to hematopoie-

sis, thereby demonstrating that self-renewal-mediated

maintenance of HSCs over time is not affected by the

absence of RB (Figure S7).

C

When 1000 freshly isolated lin�c-Kit+Sca-1+ cells were

competitively transplanted, RbD/D LKS+ displayed a

20-fold reduction in long-term repopulating potential on

a per-cell basis (Figure 4A). The reduced repopulating

potential of the RbD/D LKS+ fraction, despite a marked

increase in this phenotypic population observed in the

bone marrow (see Figure 2), demonstrates that the sur-

face phenotype of the cells does not faithfully reflect their

functional potential. We, and others, have previously

observed a lack of fidelity of phenotypic markers both in

mutant mice and following perturbation of homeostasis

in wild-type animals (Purton et al., 2006; Spangrude

et al., 1995; Tajima et al., 2000; Walkley et al., 2005).

HSCs may be lost from the BM for several reasons,

including a failed capacity of RbD/D HSCs to home and

engraft following transplantation, an increased rate of

apoptosis, or a mobilization/redistribution to extramedul-

lary sites. As cell-cycle status correlates with engraftment

capacity of HSCs (Gothot et al., 1998; Passegue et al.,

2005), we directly assessed the cell-cycle status of phe-

notypic RbD/D progenitors (LKS�), primitive progenitors

(LKS+), and HSCs (LKS+CD34�/lo). All three populations

displayed a comparable cell-cycle profile, with HSCs

from RbD/D displaying the same distribution of cells in

the G0/G1 or S phase of the cell cycle as control cells (fl/

fl: G0/G1 = 86.2 ± 5.7%; D/D: G0/G1 = 85.1 ± 2.4%, p =

0.85, fl/fl: S = 6.7 ± 1.9%; D/D: S = 7.8 ± 0.7%, p = 0.54,

n = 4 fl/fl, 7 D/D; expressed as mean ± SEM). There was

no difference in the rate of cell-cycle entry of LKS+ cells

between control and RbD/D cells as determined by

BrdU-incorporation rates at either 2 or 4 weeks post-

pIpC (Figure S9). Furthermore, analysis of the in vivo hom-

ing of RbD/D BM did not reveal a difference compared to

control BM at either 2 or 12 weeks post-pIpC (Figure 4B

and data not shown). RbD/D LKS+ cells exhibited

decreased apoptosis, as assessed by annexin-V staining,

at 2 weeks and exhibited normal levels at 12 weeks post-

pIpC compared to control LKS+ cells (Figure 4C). As we

had observed significantly increased progenitors and

HSCs in extramedullary sites, these data are consistent

with the loss of HSCs from the BM as a result of both

enhanced differentiation of HSCs within the BM and a

redistribution to extramedullary sites as a consequence

of the changes in the niche.

Myeloid-Restricted Inactivation of Rb Does

Not Result in Myeloproliferation

To determine the contribution of myeloid-derived cells

(granulocytes, macrophages, and osteoclasts) to the

myeloproliferation observed in Rb mutants, we generated

Lysozyme-M-Cre pRbfl/fl mice to achieve myeloid-

restricted deletion of Rb (Figure 5). Deletion of Rb with

Lys-M-Cre did not lead to myeloproliferation or extra-

medullary hematopoiesis, consistent with the results

(J) Number of cells of each lineage/spleen; 12 weeks post-pIpC; n R 6/genotype; *p < 0.01.

(K) Fold change in phenotypic LKS+ and LKS� in the spleen; n R 3/genotype/time point; p < 0.05.

(L) Representative spleen sections (12 weeks post-pIpC). Unless noted all data are expressed as mean ± SEM.

ell 129, 1081–1095, June 15, 2007 ª2007 Elsevier Inc. 1085

Figure 3. Increase in Progenitors in the

Bone Marrow and HSCs/Progenitors in

the Spleen of RbD/D Animals

(A) Number of day 7 CFC per femur; n R 5/ge-

notype/time point; *p < 0.05.

(B) Number of CFU-S12 from 1 3 105 whole BM

cells; n = 5/genotype.

(C) Splenic day 12 CFU-GEMM and CFU-GM/M

at 12 weeks post-pIpC; n R 9/genotype; *p <

0.01. Value inside bars is fold increase.

(D) CFU-S8/spleen; n = 5/genotype; p < 0.05.

(E) Percent PB chimerism at 17 weeks post-

transplant from either 1 3 106 or 2 3 106 whole

spleen cells from RbD/D animals 8 weeks post-

pIpC with 2 3 105 WT BM cells; n = 5/genotype.

(F) Lineage contribution of spleen-derived

HSCs at 17 weeks. Data are expressed as

mean ± SEM.

obtained by deletion of Rb from hematopoietic cells in

a wild-type environment (Figure 5A; Walkley and Orkin,

2006). We observed a subtle increase in the numbers of

granulocytes in the BM and slight reduction in erythroid

cells, but no change in either lymphoid or phenotypic

progenitor and HSC-enriched fractions (Figures 5A and

5B). Lineage distribution within the spleen or PB was

largely comparable to controls (Tables S1 and S2; data

not shown). Thus, deletion of Rb from myeloid-derived

populations does not recapitulate the phenotype observed

in the Mx-Cre model. Collectively, these results suggest

that RB may regulate HSCs and hematopoiesis in an

1086 Cell 129, 1081–1095, June 15, 2007 ª2007 Elsevier Inc.

extrinsic manner, possibly through regulating the compe-

tence of the bone marrow niche.

An Rb-Dependent Interaction between

Myeloid-Derived Cells and the BM

Microenvironment Results in Myeloproliferation

We next sought to determine the relative contributions of

the hematopoietic cells and the nonhematopoietic (non-

transplantable) elements of the BM microenvironment

to the observed phenotype. Hematopoietic cells alone

were not capable of inducing either myeloproliferation or

the loss of HSCs from the BM that we observed in the

Figure 4. Loss of HSCs from the Bone

Marrow Following Rb Deletion

(A) HSC frequency and absolute number/

femur; n = 4–5 recipients/cell dose/genotype;

experiment was performed twice; data were

pooled from two independent experiments for

calculation of HSC frequency. Primary trans-

plant data are from 6 months posttransplant.

Secondary transplant data are from 3 months

posttransplant. One thousand freshly isolated

LKS+ were transplanted, and repopulating

unit (RU) was calculated 6 months posttrans-

plant; n = 5 recipients/cell dose/genotype.

(B) Number of CFDA-SE-labeled cells in the BM

of recipients 16 hr after injection; BM from 12

weeks post-pIpC; n = 5 recipients/genotype.

(C) Apoptotic cells in the LKS+ and LKS�populations; n = 9/genotype; p < 0.05. Data

are expressed as mean ± SEM.

Mx-CrepRbD/D model (Walkley and Orkin, 2006). Consis-

tent with this conclusion we did not observe myeloproli-

feration when previously excised RbD/D HSCs were

supported by a wild-type microenvironment, even at

high cell doses (described in Figure 3).

To ascertain if RB loss from the niche was responsible

for the myeloproliferation and loss of BM HSCs, reciprocal

transplants of wild-type hematopoietic cells into lethally

irradiated Mx-Cre�pRbfl/fl and Mx-Cre+pRbfl/fl recipients

were performed, and, following establishment of hemato-

poiesis, recipients were injected with pIpC to delete Rb

from the hematopoietic microenvironment. This strategy

was successfully used to demonstrate a role for BMP

receptor type 1 in the regulation of the HSC niche (Zhang

et al., 2003), and a similar approach demonstrated that

a RARg�/� microenvironment alone could induce myelo-

proliferation (Walkley et al., 2007 [this issue of Cell]). Fol-

lowing inactivation of Rb, recipients were monitored and

analyzed at 8 and 20 weeks posttransplant. We failed to

observe significant changes in hematological parameters

of these recipients (Figure 6A). The data show that loss of

RB uniquely in either hematopoietic cells or niche cells

alone is insufficient to account for the findings in

Mx-Cre+pRbD/D mice.

Bone homeostasis is maintained through balanced

activities of mesenchymal-derived osteoblasts and

myeloid-derived osteoclasts (Martin and Sims, 2005). As

we observed a rapid loss of trabecular bone following

Mx-Cre-mediated deletion, we quantitated the numbers

of osteoclasts present in the BM and spleen. The numbers

of osteoclasts in both the BM and spleen by 6 weeks post-

pIpC were markedly increased (Figures 5C and 5D).

Osteoclasts and macrophages derived from pIpC-treated

Mx-Cre mice showed efficient deletion of Rb, as did

osteoclasts and macrophages derived from Lysozyme-

M-Cre pRbfl/fl mice (Figure 5E). Osteoclasts have been

proposed to contribute to the release of HSCs from the

bone marrow during mobilization (Kollet et al., 2006).

Having observed significantly increased neutrophils and

monocytic-derived osteoclasts in the Mx-Cre+pRbD/D

model (Figure 5C), we hypothesized that deletion of Rb

from myeloid-derived cells together with an Rb-deficient

microenvironment might recapitulate the phenotype

observed in the Mx-Cre+pRbD/D model.

Hematopoietic cells from sex-mismatched Lysozyme-

M-Cre pRbfl/fl animals were transplanted into lethally

irradiated Mx-Cre�pRbfl/fl and Mx-Cre+pRbfl/fl recipients,

and, following establishment of hematopoiesis, recipi-

ents were injected with pIpC to delete Rb from the

BM microenvironment. Analysis of Y chromosome levels

by quantitative PCR on peripheral blood leukocytes

confirmed engraftment and high-level chimerism of all

recipients prior to pIpC and at the time of analysis

(data not shown). This transplant strategy results in Rb

deficiency in myeloid-derived cells (granulocytes, mac-

rophages, and osteoclasts) and an Rb-deficient niche.

In addition, RB expression is retained within the HSC

compartment.

We observed synergistic interaction between the pRbD/D

myeloid cells and the pRbD/D microenvironment (Figure 6).

Mx-Cre+pRbD/D recipients rapidly developed signs of

distress as early as 2 weeks after the completion of

Cell 129, 1081–1095, June 15, 2007 ª2007 Elsevier Inc. 1087

Figure 5. Myeloid-Restricted Rb Deletion Does Not Result in a Myeloproliferation

(A) BM analysis; n R 6/genotype.

(B) Phenotypic primitive progenitors/femur; n R 6/genotype.

(C) Number of osteoclasts/femur; 6 weeks post-pIpC; n = 3/genotype.

(D) Number of osteoclasts/spleen; 6 weeks post-pIpC; n = 3/genotype.

(E) Rb-excision analysis of osteoclasts and macrophages derived from either Mx-Cre mutants of Lysozyme-M-Cre�pRbfl/fl mutants; n R 3/genotype.

Representative photographs of osteoclast and macrophage cultures (original magnification 103).

Data are expressed as mean ± SEM.

pIpC. No Mx-Cre+pRbD/D recipient survived beyond 11

weeks post-pIpC. Greater than 70% of recipients were

moribund by 5 weeks, in contrast to recipients of wild-

type BM cells that survived at least 20 weeks post-pIpC

1088 Cell 129, 1081–1095, June 15, 2007 ª2007 Elsevier Inc.

with no significant changes in hematopoiesis (Figure 6).

We observed rapid development of a completely penetrant

myeloproliferative disorder in the BM, characterized by

myeloid cell hyperplasia and suppression of lymphopoiesis

Figure 6. A pRb-Dependent Interaction between Myeloid Cells and the Bone Marrow Microenvironment Causes Myeloprolifera-

tion

(A) Wild-type BM was transplanted into unexcised Mx-Cre�pRbfl/fl and Mx-Cre+pRbfl/fl recipients, and 5 weeks posttransplant recipients received

pIpC. Recipients were analyzed at 8 (n = 3/genotype) and 20 weeks (n = 4/genotype) post-pIpC. Data are expressed as fold change of Mx+ recipients

compared to Mx�Cre�pRbfl/fl recipients (normalized to 100%). Data are shown from 8 weeks post-pIpC (comparable results at 20 weeks).

(B) Sex-mismatched Lysozyme-M-Cre+pRbfl/fl BM was transplanted into unexcised Mx-Cre�pRbfl/fl and Mx-Cre+pRbfl/fl recipients, and 5 weeks post-

transplant pIpC was administered. Data are expressed as fold change of Mx+ recipients compared to Mx-Cre�pRbfl/fl recipients (normalized to 100%).

y = approximate time of analysis post-pIpC; n = 11 Mx-Cre+ recipients in three independent experiments (two found dead 2 weeks post-pIpC); n = 10

Mx-Cre� control recipients.

(C) Comparison of spleen weights amongst groups.

(D) Splenic hematopoiesis in Mx-Cre+pRbfl/fl recipients of Lysozyme-M-Cre+pRbfl/fl bone marrow. Data are expressed as fold change compared to

Mx-Cre�pRbfl/fl recipients (normalized to 100%).

and erythropoiesis. Splenomegaly, accompanied by mye-

loid and erythroid hyperplasia and extramedullary hema-

topoiesis, was also observed, demonstrating a striking

similarity to the full Mx-Cre model (see Figure 2 and

Tables S1 and S2). The increased spleen size of Mx-

Cre� recipients of LysM-Cre pRbfl/fl BM can be accounted

for by the LysM-Cre pRbfl/fl BM itself, rather than a

contribution from the recipient environment (Tables S1

Cell 129, 1081–1095, June 15, 2007 ª2007 Elsevier Inc. 1089

and S2). These data demonstrate that the observed mye-

loproliferation is the consequence of an RB-dependent

interaction between myeloid-derived cells and the BM

microenvironment and that it develops independent of the

HSC RB status. Our data provide direct experimental

evidence that myeloproliferation may ensue from aberrant

interactions between myeloid-derived cells and the BM

microenvironment, revealing hematopoietic extrinsic con-

tribution to myeloproliferation.

DISCUSSION

We sought to determine the role RB plays in the regulation

of hematopoiesis and stem cell function. Recent studies

suggest that cell-cycle regulation is an important determi-

nant of stem cell fate; however, none have discriminated

between intrinsic or extrinsic contributions (Cheng et al.,

2000; Janzen et al., 2006; Walkley et al., 2005; Yuan

et al., 2004). RB was implicated as an important regulator

of stem cell maintenance in Arabidopsis; however, the

limitations of the experimental system did not allow for

the clear demonstration of a stem cell intrinsic role for

RB (Wildwater et al., 2005). Here we demonstrate that

RB extrinsically regulates HSCs by maintaining the com-

petence of the adult bone marrow to support HSCs and,

in turn, normal homeostatic hematopoiesis.

Rb and Stem Cell Self-Renewal

Understanding the regulation of cell cycle in stem cells is

important from several perspectives. Stem cells must en-

ter the cell cycle to self-renew; hence, induction of cycling

may be desirable to achieve HSC expansion. Engraftment

of transplanted HSCs is cell cycle dependent (Gothot

et al., 1998; Passegue et al., 2005). The slow cycling of

HSCs may spare them from acute toxicity (such as che-

motherapy) but may also prevent neoplastic cells from

eradication (Hodgson and Bradley, 1979; Lerner and

Harrison, 1990). Our understanding of the normal regula-

tion of self-renewal will also provide insight into tumori-

genesis, where self-renewal pathways are thought to be

active (Krivtsov et al., 2006).

The importance of cell-cycle regulation in HSC fate

decisions has been suggested by the analysis of animals

deficient in negative cell-cycle regulators such as

p21Cip1, p27Kip1, and p16INK4a/p19ARF (Cheng et al.,

2000; Stepanova and Sorrentino, 2005; Walkley et al.,

2005). However, these studies have not revealed if such

HSC defects are cell intrinsic or extrinsic in nature. The

‘‘Rb pathway’’ has also been implicated in phenotypes ob-

served in both the Bmi1�/� and ATM�/� HSCs (Ito et al.,

2004; Lessard and Sauvageau, 2003; Park et al., 2003).

Surprisingly, we did not observe an intrinsic requirement

for Rb in HSCs. If provided with a wild-type niche, RbD/D

HSCs contribute normally to multilineage hematopoiesis

and display serial transplant potential comparable to

wild-type HSCs. Furthermore, we failed to detect alter-

ations in numerous cell-cycle- or self-renewal-associated

genes in Rb-deficient HSCs and progenitors isolated from

1090 Cell 129, 1081–1095, June 15, 2007 ª2007 Elsevier Inc.

both a wild-type and mutant microenvironment, consis-

tent with our interpretation that Rb is dispensable in the

HSCs (Figure S8). Our observations, taken together with

those from analysis of p27Kip1 mutant mice, reveal that

cell-cycle regulation is a novel extrinsic regulator of hema-

topoiesis (Chien et al., 2006; Walkley and Orkin, 2006). The

loss of BM HSCs in the Mx-Cre model is a secondary

consequence of the disrupted environment within the

BM and was not observed in the context of a wild-type

niche, demonstrating that myeloproliferative-like disor-

ders may deplete HSCs from the BM. A reanalysis of

cell-cycle mutants proposed to harbor HSC defects is

needed to clarify the intrinsic and extrinsic roles that

cell-cycle regulation plays in these phenotypes.

It has been documented that self-renewal of embryonic

stem cells occurs in an RB independent manner (Stead

et al., 2002); however, the RB pathway is thought to be

near universally targeted in human cancer cells (Hanahan

and Weinberg, 2000). We have described that self-renewal

of nontransformed HSCs occurs independent of RB, high-

lighting the important question of what role RB plays in the

regulation of the process of cellular self-renewal in both

normal and oncogenic settings. It may be that the require-

ment for RB and the RB pathway in self-renewal is devel-

opmentally and lineage dependent, with progenitor cells

having a greater dependence on RB for their division

than bona fide stem cells. One prediction of such a hypoth-

esis is that mutation of the RB pathway is of greater benefit

to a progenitor cell than a stem cell during tumor formation.

Rb and Hematopoiesis

Previous studies examining the role of RB in hematopoie-

sis have raised conflicting evidence regarding intrinsic and

extrinsic effects, particularly in erythropoiesis (Clark et al.,

2004; Iavarone et al., 2004; Spike et al., 2004; Whyatt

and Grosveld, 2002). Our study utilized compartment-

restricted somatic mutagenesis to analyze the role of

RB in adult hematopoiesis and HSCs. Compartment

restricted deletion enables a direct assessment of the

contribution of hematopoietic and nonhematopoietic cells

to the observed phenotype. We have not observed a

progressive failure of hematopoiesis as reported by Spike

et al. (2004) when RB-deficient HSCs were supported by

a wild-type environment, nor was this observed in a

separate study utilizing germline-deficient fetal liver

hematopoietic cells (Hu et al., 1997). In contrast to in vitro

findings (Iavarone et al., 2004), deletion of Rb from

myeloid-derived cells using Lys-M-Cre did not result in

anemia in vivo. Further studies utilizing lineage-restricted

deletion of Rb will be required to clarify the role of RB in

erythropoiesis.

Interactions between Hematopoietic Cells

and Their Microenvironment Regulate HSCs

and Hematopoiesis

The phenotype of Mx-Cre+pRbD/D animals is due to an

RB-dependent interaction between myeloid-derived cells

(most probably macrophages and osteoclasts) and the

Table 1. Summary of the Phenotype Observed Following Loss of Rb

Bone Marrow

Condition Hematopoietic CellsNiche /Microenvironment HSC Myeloid Lymphoid Erythroid

Mx-Cre pRbfl/fl model D/D D/D YYY [[[ Y 4

D/D HSC into WT nichea D/Db WT 4 [ 4 4

WT HSC into D/D niche WT D/Db 4 4 4 4

D/D myeloid cells D/D (myeloid) WT 4 4 4 4

D/D myeloid

cells into D/D niche

D/D (myeloid) D/Db [[[ Y Y

Spleen and Extramedullary Sites

Condition Hematopoietic Cells

Niche /

Microenvironment HSC / Progenitorsc Myeloid Lymphoid Erythroid

Mx-Cre pRbfl/fl model D/D D/D [[[ [[[ 4 [[[

D/D HSC into WT nichea D/Db WT 4 4 4 4

WT HSC into D/D niche WT D/Db 4 4 4 4

D/D myeloid cells D/D (myeloid) WT 4 4 4 [

D/D myeloid

cells into D/D niche

D/D (myeloid) D/Db [[[ [[[ 4 [[[

a Summary of data previously described (Walkley and Orkin, 2006).b Indicated compartment was nondeleted (Mx-Cre pRbfl/fl) at time of transplant and deleted 5 weeks after transplant with pIpC

injection.c HSC and Progenitors as determined by flow cytometry (Lin�c-Kit+Sca-1+ and Lin�c-Kit+Sca-1�) and in vitro progenitor analysis.

bone marrow microenvironment (summarized in Tables 1

and S1). Evidence supports a direct role for the bone mar-

row microenvironment, but we cannot entirely exclude

a contribution from other sites of Cre activity in the Mx-

Cre model. Myeloproliferation is generally considered to

be hematopoietic intrinsic, and evidence from the overex-

pression of activated kinase receptors in mouse models is

consistent with this view (Araki et al., 2004; Chan et al.,

2004; Le et al., 2004). In light of the data derived from

Mx-Cre+pRbD/D mice, the BM microenvironment may

play an active role in the promotion and/or maintenance

of myeloproliferative disorders. Additional studies are

required to define the cell(s) within the BM niche that are

responsible for this interaction. The BM microenvironment

is composed of numerous nonhematopoietic cell types

including osteoblasts, endothelial cells, adipocytes, and

nerve cells. Histomorphometry demonstrated a significant

disruption to bone homeostasis in the Rb-deficient

animals, correlating with the observed mobilization and

extramedullary hematopoiesis (Figures 2E–2H). Myelo-

proliferation in Mx-Cre+pRbD/D mice is dependent on con-

comitant deletion of Rb from both myeloid-derived cells

and the environment. In other situations, myeloprolifera-

tion may result directly from an aberrant niche and may

be independent on mutation(s) within hematopoietic cells

(Walkley et al., 2007).

Evidence of the role of stroma and the microenviron-

ment in oncogenesis is accumulating, notably from analy-

sis of solid tumors. Moreover, mathematical modeling of

tumor behavior predicts that the environment is a major

selective modifier of tumor morphology and phenotype

(Allinen et al., 2004; Anderson et al., 2006; Balkwill,

2004; Hill et al., 2005; Kurose et al., 2002; Oh et al.,

2004; St Croix et al., 2000). Somatic mutations divergent

from those found in the tumor have been identified in stro-

mal cells. In prostate cancer, results suggest that such

mutations may contribute nonautonomously to tumor

behavior (Hill et al., 2005; Kurose et al., 2002). Under-

standing the interactions between hematopoietic cells

and their microenvironment is directly relevant to hemato-

poietic disease. Mutations in the Rb pathway occur in

�75% of cases of multiple myeloma (Kramer et al.,

2002). Multiple myeloma clearly demonstrates that the

interaction of hematopoietic cells—in this case B cells—

and the BM microenvironment is a major contributor

to disease (Hideshima and Anderson, 2002; Mitsiades

et al., 2006). These studies have focused on mutations

present in established disease but have not addressed

the role of the microenvironment or stroma in the initiation

of the disease process.

In addition to our data focused on RB loss, the signifi-

cance of the hematopoietic microenvironment to disease

initiation has been suggested by recent studies. Mx-

Cre+PtenD/D mice (Pten-deficient hematopoietic cells

and microenvironment) develop rapid and aggressive

myeloproliferation that progresses to overt leukemia/

lymphoma in 4 to 5 weeks postdeletion (Yilmaz et al.,

2006; Zhang et al., 2006b). However, when Pten deletion

Cell 129, 1081–1095, June 15, 2007 ª2007 Elsevier Inc. 1091

was activated in the context of a wild-type BM microenvi-

ronment, phenotypic and functional HSCs were lost

without evidence of myeloproliferation or transformation

(Yilmaz et al., 2006). This striking result suggests that

PtenD/D hematopoietic cells alone are not intrinsically sus-

ceptible to myeloproliferation and subsequent malignant

transformation in the presence of a wild-type microenvi-

ronment. Mutations in PTEN have been reported in the

stroma of human breast tumors, suggesting a broader

role for this pathway in the microenvironment and stroma

of diverse organ systems (Kurose et al., 2002). Intriguingly

JunB, Bmi-1, and ATM, implicated in HSC regulation and

myeloproliferation, also have roles in regulating the bone

marrow microenvironment (Kenner et al., 2004; Oguro

et al., 2006; Passegue et al., 2004; Rasheed et al.,

2006). JunB-deficient mice develop severe osteopenia

due to intrinsic defects in osteoclasts and osteoblasts,

cellular constitutents of the HSC niche, while ATM

mutants develop osteoporosis as a result of defective

osteoblast differentiation. The contribution of these micro-

environmental defects to the HSC phenotypes in these

mutants has yet to be described. Such results demon-

strate the need for further analysis of the interaction

between the hematopoietic cells and their environment.

This reconsideration will further our understanding of

normal homeostatic hematopoiesis and the development

of hematopoietic disease.

Our finding that the myeloproliferative-like disorder in

the Rb mutants is the result of an interaction between

myeloid-derived cells and the bone marrow microenvi-

ronment, together with the microenvironment-induced

myeloproliferative-like disorder that develops in the

RARg�/�mice (Walkley et al., 2007), underscores a previ-

ously unrecognized role for the hematopoietic microenvi-

ronment in the development of myeloid disease. These

data further suggest that mutations within the hematopoi-

etic niche might also serve as initiating events in the devel-

opment of hematopoietic disease. In contrast to previous

reports of the importance of cell-cycle regulation in HSC

fate determination, we find scant evidence for an intrinsic

requirement for RB in HSCs and that, indeed, RB is a novel

extrinsic regulator of hematopoietic stem cells. As our

findings underscore, interactions between hematopoietic

cells and the bone marrow niche/microenvironment pro-

foundly affect hematopoietic homeostasis and the behav-

ior of HSCs.

EXPERIMENTAL PROCEDURES

A detailed version of the Experimental Procedures can be found in the

Supplemental Data.

Experimental Animals

pRbfl/fl mutant mice were generously provided by Dr. Tyler Jacks

(Massachusetts Institute of Technology, MA, USA; Sage et al., 2003).

Mx-Cre transgenic mice have been previously described (Kuhn et al.,

1995; Walkley and Orkin, 2006). Lys-M-Cre animals were purchased

from The Jackson Laboratory (Clausen et al., 1999). All experiments

1092 Cell 129, 1081–1095, June 15, 2007 ª2007 Elsevier Inc.

were performed with approval of the respective Institute Animal Ethics

Committees (DFCI or CHB).

Flow Cytometry Analysis

All antibodies and clone numbers are listed in Supplemental Experi-

mental Procedures. Flow cytometry was performed on a FACSCalibur,

and sorting was performed on a FACS Aria; all data were analyzed

using Cell Quest Pro software (Becton Dickinson).

Progenitor Cells Assays: CFC and CFU-S

BM, spleen cells, and PB leukocytes were assessed for in vitro colony-

forming cell (CFC, defined as >50 cells/colony) potential at either day 7

CFC (DME/agar media) or day 12 CFC (IMDM/methylcellulose media)

as described in Supplemental Experimental Procedures. Colony-form-

ing unit-spleen (CFU-S) was performed using the CFU-S assay of Till

and McCulloch with both WBM (CFU-S day 12)- and spleen (CFU-S

day 8)-derived cells (Purton et al., 1999; Till and McCulloch, 1961).

Long-Term Repopulating Cell (HSC) Analysis

Limit-dilution competitive repopulation analysis was performed as

previously described (Purton et al., 2006; Szilvassy et al., 1990; Walk-

ley et al., 2005) using test cell doses of 5 3 104, 2 3 105, and 2 3 106

cells competed against 2 3 105 WT WBM (CD45.1+/CD45.2+). Four to

five recipients/cell dose/genotype/experiment were transplanted, and

the experiment was performed in duplicate. Recipients were analyzed

at 3 and 6 months posttransplant. HSC frequencies were calculated

using L-Calc software (StemCell Technologies Inc.) using Poisson sta-

tistical analysis (Taswell, 1981). Secondary recipients were analyzed at

3 months posttransplant. One thousand freshly isolated LKS+ were in-

jected with 2 3 105 competitor WBM into five recipients per genotype.

Whole spleen cells were isolated from RbD/D 8 weeks post-pIpC and

competitively transplanted (1 3 106 or 2 3 106) with 2 3 105 competitor

WBM into five recipients per genotype.

For transplant of WT or Lys-M-Cre+pRbfl/fl WBM into Mx-Cre+ or Mx-

Cre�pRbfl/fl recipients, recipients were transplanted with 3 3 106 WBM

from sex mismatched animals. Chimerism was confirmed either by

CD45.1/CD45.2 allele analysis or by Y chromosome qPCR as

indicated.

Analysis of Transplant Recipients

PB from each individual recipient was obtained from the retro-orbital

plexus at the indicated time points posttransplant and was analyzed

as described for chimerism and lineage contribution of test cells

(Purton et al., 2000; Walkley et al., 2005).

Statistical Analyses

Statistical analyses were performed using the paired and unpaired

Student’s t test. Calculation of HSC frequency was performed using

Poisson statistical analysis using L-Calc software (StemCell Technol-

ogies Inc.). Histomorphometric data was analyzed by ANOVA followed

by Fisher’s PLSD Test.

Supplemental Data

Supplemental Data include Experimental Procedures, References,

two tables, and nine figures and can be found with this article online

at http://www.cell.com/cgi/content/full/129/6/1081/DC1/.

ACKNOWLEDGMENTS

The authors thank Tyler Jacks for the generous provision of pRbfl/fl

mice and Ingrid Poulton for bone histology. We thank David Williams,

Kevin Shannon, Jack Martin, Hans Widlund, and Ernestina Schipani for

discussion, helpful suggestions, and critical comment; DFCI and

Children’s Animal Facility Staff for care of experimental animals;

John Daley and Suzan Lazo-Kallanian of DFCI HemNeo Flow facility

for assistance with FACS sorting; David Dombkowski for assistance

with FACS analysis; and DFCI/Harvard Cancer Centre Rodent Histol-

ogy Core.

This work was supported in part by a Center of Excellence in Molec-

ular Hematology Award from the NIH-NIDDK (S.H.O). C.R.W is a Spe-

cial Fellow of the Leukemia & Lymphoma Society, and S.H.O is an In-

vestigator of the Howard Hughes Medical Institute.

C.R.W designed and performed experiments, analyzed and inter-

preted data, and wrote the paper; J.M.S performed experiments;

N.A.S performed experiments and interpreted data; L.E.P analyzed

and interpreted data; S.H.O. analyzed and interpreted data and wrote

the paper.

Received: November 14, 2006

Revised: February 15, 2007

Accepted: March 29, 2007

Published: June 14, 2007

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