doi:10.1182/blood-2005-02-0685Prepublished online October 18, 2005;   

 Hui Yu, Youzhong Yuan, Hongmei Shen and Tao Cheng p21Cip1/Waf1 in opposite mannersHematopoietic stem cell exhaustion impacted by p18INK4C and

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Hematopoietic stem cell exhaustion impacted by p18INK4C and p21Cip1/Waf1 in opposite manners

Hui Yu 1,2,3, Youzhong Yuan1,2,3, Hongmei Shen1,2, Tao Cheng1,2,4 1Department of Radiation Oncology, University of Pittsburgh School of Medicine and

2Stem Cell Biology Laboratory, University of Pittsburgh Cancer Institute, Pittsburgh PA 15213 3These authors contributed equally to this work. 4Correspondence to: Tao Cheng, MD Hillman Cancer Center Research Pavilion 5117 Center Ave, Room 2.42e Pittsburgh, PA. 15213-1863 Tel: 412-623-3249 Fax: 412-623-7778 E-mail: chengt@upmc.edu Key words: hematopoietic stem cell, self-renewal, exhaustion, aging, transplantation, cell cycle regulators Running title: Stem cell exhaustion Word counts: Abstract-204; Main body (including methods)-3825; Methods-854; Display-4 figures and 1 table; Legends-1124.

Blood First Edition Paper, prepublished online October 18, 2005; DOI 10.1182/blood-2005-02-0685

Copyright © 2005 American Society of Hematology

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Abstract: Transplantation-associated stress can compromise the hematopoietic potential of

hematopoietic stem cells (HSCs). As a consequence, HSCs may undergo so called

“exhaustion” in serial transplant recipients, for which the cellular and molecular bases are not

well understood. Hematopoietic exhaustion appears to be accelerated in the absence of

p21Cip1/Waf1 (p21), a cyclin-dependent kinase inhibitor (CKI) in irradiated hosts. Our recent

study demonstrated that unlike loss of p21, deletion of p18 INK4C (p18), a distinct CKI, results

in improved long-term engraftment largely owing to increased self-renewing divisions of HSCs

in vivo. We show here that HSCs deficient in p18 sustained their competitiveness to wild-type

HSCs from unmanipulated young mice, and retained multilineage differentiation potential after

multiple rounds of serial bone marrow transfer over a period of more than 3 years. Further,

p18 absence significantly decelerated hematopoietic exhaustion caused by p21 deficiency.

Such an effect was shown to occur at the stem cell level, likely by a counteracting mechanism

against the cellular senescence outcome. Our current study provides new insights into the

distinct impacts of these cell cycle regulators on HSC exhaustion and possibly HSC aging as

well under proliferative stress, thereby offering potential pharmacological targets for

sustaining the durability of stressed HSCs in transplant or elderly patients.

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Introduction

Hematopoietic stem cells (HSCs) are the most important element for establishing the long-term

engraftment of hematopoietic transplants in recipients. Thus, the sustained self-renewal potential of

donor HSCs is critical for maintaining the long-term durability of the graft. While HSCs are thought to

be capable of self-regeneration in vivo over a lifetime without an apparent limit under homeostatic

conditions,1,2 it is well known that the repopulating ability of HSCs can be significantly compromised in

transplant recipients.3-8 Studies by several laboratories have demonstrated that the functional HSC

units reach only 4-10% of normal levels after each transplantation4,7-10 and as a consequence, serial

bone marrow transfer can only sustain hematopoiesis for 4-6 rounds in irradiated mouse recipients.

The limited repopulating ability of HSCs during serial transplantation has led to one view that the self-

renewal ability of HSCs is intrinsically limited.11 But extrinsic factors such as the transplantation

procedure4 and the irradiated bone marrow microenvironment7 are also likely involved. Since a

substantial portion of stem cell transplants still involve the use of total body irradiation (TBI) (though

not with an ablative dose nowadays) as a host conditioning regime, it is of clinical relevance to define

an effective approach for sustaining the self-renewal potential of transplanted HSCs in the irradiated

recipients. Despite the importance of this issue, the potential exhaustion of donor HSCs in transplant

recipients has not been resolved at the molecular level.

Given the limited success of in vitro HSC expansion to date, in vivo manipulations of HSCs appear to

be important alternatives to enhance HSC therapy. In addition to the administration of hematopoietic

growth factors2 and the alteration of homing receptors (such as CXCR4 and CD26)12,13 or micro-

environmental elements (such as the stem cell “niche”),14 direct manipulation of the intracellular self-

renewal machinery is an important strategy currently being explored. Cell cycle regulators have been

demonstrated to be intrinsically involved in the self-renewal of adult stem cells.15 In particular, cyclin-

dependent kinase inhibitors (CKIs) appear to have an important role in modulating the self-renewing

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potential of stem cells. Interestingly, different CKIs appear to affect self-renewal in distinct manners.

For instance, in the absence of p21Cip1/Waf1 (p21 hereafter), the founding member of CKIs, the

hematopoietic potential of HSCs was nearly exhausted after two rounds of bone marrow

transplantation.10 In contrast, we have recently documented an engraftment advantage of the

hematopoietic cells deficient in p18INK4C (p18 hereafter), a member of the INK4 family of CKIs.16 This

advantage appears to be largely if not solely due to increased self-renewal of transplanted HSCs in

vivo16 rather than a general increase in proliferation of the donor cells (Shen et al, manuscript in

preparation).

Based on our previous results, we sought to examine whether the known exhaustion of transplanted

HSCs in irradiated hosts can be significantly overcome or minimized by suppressing p18 expression

during prolonged long-term engraftment. To this end, we have focused on the regenerative ability of

transplanted p18-/- HSCs, in comparison with that of HSCs deficient in p21 or in both p18 and p21,

following serial bone marrow transplants over an extended time period. Our current study

demonstrates that deleting p18 in HSCs is effective in sustaining the durability of transplanted HSCs

beyond the lifetime of a mouse, and this deletion is able to substantially compensate for the

deleterious effect of p21 deletion on hematopoietic repopulation, likely due to the opposite effects of

p18 and p21 on HSC senescence under stress conditions.

Materials and Methods

Mice

p18+/- mice and p18-/-p21-/- double knockout mice in C57BL/6;129/Sv background were kindly provided by David

Franklin at Purdue University (p18+/- mice were originally created by Franklin et al in Yue Xiong’s laboratory17).

p18-/- and p18+/+ littermates were produced from p18+/- breeding pairs that are maintained in our laboratory.

p21-/- mice and p21+/+ littermates were kindly provided by Andrew Stewart at University of Pittsburgh (the p21+/-

breeding mice in C57BL/6;129/Sv background were purchased from the Jackson Laboratories [Bar Harbor, ME]

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and were originally created in Tyler Jacks’ laboratory18). Female C57BL/6129/SvF1 recipients at the age of 2

months were purchased from the Jackson Laboratories. All the procedures involved in the mouse work were

approved by the Institutional Animal Care and Use Committee of University of Pittsburgh.

Serial bone marrow transplantation (sBMT)

Bone marrow nucleated cells (BMNCs) (2 x 106/host) from male p18+/+ or p18-/- donor mice at the age of 2

months were transplanted into lethally irradiated (10 Gy) female wild-type recipients (2 months old). The same

procedure was applied in secondary, tertiary and quaternary recipients at intervals as indicated in Figure 1A.

Three animals in each group were sacrificed after each sBMT for measuring the cobble-stone area forming cell

(CAFC) and colony forming cell (CFC) in bone marrow as previously described.10,16 Blood was collected 12

months after the 4th round of sBMT to measure the donor contribution with the semi-quantitative PCR for Y-

chromosome.10,16

Competitive bone marrow transplantation (cBMT) coupled with serial transfer

The cBMT procedure was detailed in our previous publication16 and is further illustrated in Figure 2A. Briefly,

test cells (2 x 106 BMNCs/host) from p18-/- or p18-/-p21-/- male mice and an equal number of wild-type BMNCs

(competitor cells) from male mice (wild-type litter mates in primary cBMT and then wild-type C57BL/6129/SvF1

mice of 2 months old in subsequent rounds of cBMT) were co-transplanted into lethally-irradiated (10 Gy)

female recipients. The competitor cells from unmanipulated marrow were repetitively used in each sequential

cBMT as a standard measure for determining the repopulation ability of varied types of the original input cells

over the course of the serial transfer. Blood was collected after each cBMT and the relative contribution of test

cells (-/-) to the competitor cells (+/+) in the reconstituted host was quantified by the semi-quantitative PCR as

previously described.16 Some mice were sacrificed after each cBMT for the genotypic analyses in bulk BMNCs,

different cell lineages, and different hematopoietic compartments by the PCR described below.

Flow cytometric analysis and cell sorting

One of the well-characterized phenotypes for murine HSC, CD34-Lin-c-Kit+Sca-1+(CD34-LKS)19, was applied in

our current study. The method used to quantify the frequency of CD34-LKS in bone marrow was previously

described.16 To isolate the cells, Sca-1+ cells were enriched with the EasySep Kit (StemCell

Technologies,Vancouver, BC, Canada) according to the manufacturer’s protocol. The cells were stained with a

mixture of biotinylated antibodies against mouse CD3, CD4, CD8, B220, Gr-1, Mac-1 and TER-119 (Caltag,

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Burlingame, CA), then co-stained with streptavidin-PE-Cy7, anti-Sca-1-PE, anti-c-Kit-APC and anti-CD34-FITC

(BD Pharmingen, San Diego, CA). Propidium iodide was used for dead cell exclusion. CD34-LKS cells were

sorted into 96-well plates (Becton Dickinson, Franklin Lakes, NJ) at one cell per well using the MoFlo High-

Speed Cell Sorter with subsystems of Cyclone Automated Cloner and Sort Master Droplet Control. For sorting

the mature cells in different lineages, BMNCs were stained with a mixture of anti-CD3-PE, anti-Gr-1-PE-Cy7,

anti-Mac-1-APC and anti-B220-PE-Texas Red (BD Pharmingen). Cells of different lineages including T (CD3+),

B (B220+), and myeloid (Mac-1+) were sorted into separate tubes and then lysed for genotyping by PCR

analysis.

Clonal cell culture

Single CD34-LKS cells were deposited into 96-well plates (one cell per well). Each well contained 100µl of

IMDM supplemented with 50 ng/ml of Flt3 ligand (Flt3-L), 50ng/ml of SCF, 10ng/ml of TPO and 10ng/ml of IL-3

(PeproTech, Rocky Hill, NJ). The colony forming cell (CFC) assay with bulk BMNCs was performed according

to the manufacturer’s instructions (Stem Cell Technologies). 7~10 days after initiation of these clonal cultures,

sufficient colonies were harvested under a microscope and lysed for subsequent PCR reaction.

PCR for tracing donor contribution

Primer sequences for amplifying the p18 and p21 mutant/wild-type alleles were described in our previous

publications.10,16 In addition to the genotyping PCR for p18, the following primer pair was used to confirm the

absence of p21 in p18/p21 double mutant cells: p21null-F (5’-GCG AGG ATC TCG TCG TGA C-3’), and p21

null-R (5’-TCA TCA ATT TAT GCA GAC-3’). In the PCR for quantifying the Y-chromosome DNA (Sry), the

following primers were used: Sry-F (5’-TGG GAC TGG TGA CAA TTG TC-3’), Sry-R (5’-GAG TAC AGG TGT

GCA GCT CT), Myo-F (5’-TTA CGT CCA TCG TGG ACA GC-3’) and Myo-R (5’-TGG GCT GGG TGT TAG

TCT TA-3’). Nucleated blood cells were lysed in 1X PCR buffer containing 2.5mM MgCl2 and 20µg/ml

Proteinase K for 1 hour at 60°C, followed by an inactivation of the reaction for 20 min at 95°C. The parameters

for thermal cycling of PCR were as follows: 30s at 94°C, 30s at 57°C and 1 min at 72°C (30 cycles for the semi-

quantitative analysis with bulk blood cells and 35 cycles for the single colony analysis).

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Results

In vitro activity of p18-/- hematopoietic cells from the serially-transplanted recipients We first examined the exhausting effect of irradiated hosts on transplanted HSCs with the classical

serial bone marrow transplantation (sBMT), in which no competitor cells were co-transplanted (Figure

1A). Bone marrow nucleated cells (BMNCs) from male p18+/+ or p18-/- mice were transplanted into

lethally-irradiated female mice and sBMT was sequentially performed for 4 rounds with an interval of

3-4 months. During each sBMT, the in vitro activity of both HSCs and hematopoietic progenitor cells

(HPCs) from p18-/- recipient mice was significantly higher than that from p18+/+ transplanted mice, as

examined by the in vitro surrogate assays for HSCs and HPCs, namely the cobblestone-area forming

cell (CAFC) and colony forming cell (CFC), respectively (Figure 1B). Of note, CAFC frequency at day

35 in p18-/- transplanted mice was five times higher than that in p18+/+ transplanted mice after

secondary sBMT. CFC frequency was maintained at a normal level (that of unmanipulated bone

marrow) in p18-/- transplanted mice while it gradually declined in p18+/+ transplanted mice during 3

rounds of sBMT. These results contrast with the accelerated hematopoietic exhaustion in the

absence of p21 during sBMT, as shown in a previous study.10

Sustained in vivo hematopoiesis of p18-/- HSCs after multiple BMTs

Our next step was to determine the in vivo long-term repopulating ability (LTRA) of HSCs 12 months

after quaternary sBMT, by quantifying the presence of HSC progeny in blood (Figure 1A). PCR-

based semi-quantitative analysis for the Y chromosome-specific sequence (Sry) was used to track

cellular origin.10 Based on the standardization simultaneously generated under identical PCR

conditions, we calculated the percentage of donor cell contribution to recipient hematopoiesis. As we

expected, most recipients (4 of 6) of p18+/+ HSCs had undetectable levels of donor cells in their blood

(Figure 1C). In contrast, the original p18-/- male donor-derived cells accounted for 66.7% on average

(60-73%) of whole mature blood cells in all recipients (N=7). By an analysis with the single limiting

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dose, we estimated that there was only one HSC unit (being able to give rise to detectable donor cells

in the blood of a recipient animal) per 5 x 106 BMNCs in p18+/+ transplanted recipients after 4 rounds

of sBMT in contrast to one HSC per 1-2 x 105 BMNCs in normal unmanipulated marrow.20 The 100%

engraftment rate in the p18-/- transplanted group precluded us from directly estimating the frequency

of functional units of HSCs in the p18-/- recipients. However, p18-/- HSCs at this later point could be

estimated following serial competitive bone marrow transplantation (cBMT) for three times over 30

months, subsequent to our previous study (Figure 2A).16

In the cBMT experiment, equal numbers of p18-/- BMNCs and competitor cells (wild-type BMNCs

freshly isolated from untransplanted mice) were co-transplanted into lethally-irradiated recipients in

each cBMT. Because LTRA and HSC frequency in mouse wild-type BMNCs have been well

documented, the fixed number (2 x 106) of unmanipulated competitor cells can serve as a standard

measure for varied types of input cells to be compared (Figure 2A). Blood was collected 6-14 months

after each cBMT and the relative contribution to hematopoiesis from each genotype (p18-/- versus

wild-type) was quantified by the semi-quantitative PCR assay.16 After 3 rounds of cBMT in 30

months, LTRA of p18-/- HSCs was still equivalent (54.9%) to that of the competitor cells (Figure 2B

and Figure 2C), which indicates there is no apparent exhaustion of hematopoietic potential as

compared to that of the wild-type transplanted cells at this time point.

An opposing effect of p18 absence over p21 absence on HSC exhaustion

To test whether p18 absence can counteract the known accelerated hematopoietic exhaustion

caused by p21 absence,10 we directly assessed the long-term engraftment of hematopoietic cells

from mice deficient in both p21 and p18 (p18-/-p21-/-, hereafter, abbreviated for the double-

knockout).21 LTRA of HSCs from p18-/-p21-/- mice was compared to that from p18-/- mice using the

cBMT assay (Figure 2A).16 In contrast to a previous study showing early exhaustion of hematopoietic

reconstitution in the absence of p21 after sBMT,10 p18-null was able to overcome the accelerated

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hematopoietic exhaustion caused by p21 deletion as demonstrated by 51.1% and 34.1% of

reconstitution level in secondary and tertiary recipients of the p18-/-p21-/- cells, respectively (Figure 2B

and Figure 2C). As a confirmatory result to the previous report10, we were also able to observe an

accelerated hematopoietic exhaustion in a different experiment where p21-/- BMNCs were co-

transplanted with the same competitor cells in the current study (Figure 2D). The fact that p18-/-p21-/-

HSCs were able to sustain their LTRA after 3 rounds of cBMT suggests that the absence of p18 can

significantly mitigate the negative impact of p21 deletion on the LTRA of HSCs. Therefore, these

data provide direct evidence for the opposite effects of p18 and p21 on the hematopoietic potential of

HSCs in the context of serial transplantation.

Multilineage differentiation of p18-/- or p18-/-p21-/- HSCs

Multilineage analysis of bone marrow cells from tertiary recipients was performed to determine

whether p18-/- or p18-/-p21-/- HSCs maintained their ability to give rise to both myeloid and lymphoid

lineages after multiple cBMTs performed over a prolonged period of time. The

immunophenotypically-defined myeloid (granulocytes and monocytes) and lymphoid (T and B

lymphocytes) cells were sorted for the determination of p18 genotype (p18-/- vs. p18+/+). As shown in

Figure 3, substantial engraftment levels of p18-/- cells were seen in both myeloid and lymphoid

lineages, indicating the multilineage potential of transplanted p18-/- or p18-/-p21-/- HSCs for 30 months

over multiple cBMTs (at the cellular age of 36 months). Notably, the relative contribution of p18-/-

donor cells to competitor cells in myeloid lineage was higher than that observed in lymphoid lineage

(Figure 3B). This result is in agreement with previous studies by others that showed favored

myelopoiesis over lymphopoiesis in aged mice, which is indicative of ongoing aging of hematopoietic

cells in vivo.22-24 However, the relative decrease of T-lymphopoiesis was significantly improved in the

p18-/- group in comparison with that in the p18-/-p21-/- group (p < 0.05), suggesting distinct roles of p18

and p21 in T-lymphopoiesis in addition to their direct effects on HSCs.

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Representation of p18-/-, p18-/-p21-/- or p21-/- genotype in the HSC compartment

To further distinguish the contribution of test cells versus competitor cells in the stem cell

compartment, we isolated HSCs with the primitive immunophenotype for murine HSCs, CD34-LKS,19

from the recipients of cBMT and determined their genotype at the single cell level. It should be noted

that this immunophenotype with LTRA does not seem to change in aged or transplanted mice based

on the data by others24 and our own unpublished observation. Single HSC cells were sorted into 96-

well plates (one cell per well) and cultured in medium with appropriate cytokine stimuli to form

colonies (more than 30 cells/colony) in 7-10 days, which allowed us to efficiently track the origin of

individual HSCs that were initially isolated from the marrow. Sufficient numbers of colonies were

randomly picked up for the genotyping PCR analysis on the marrow harvested at varied time points

after cBMT (Table 1). Data demonstrated that the majority of CD34-LKS cells were of p18-/- or p18-/-

p21-/- origin after tertiary or quaternary cBMT. Interestingly, the dominance of p18-/-p21-/- genotype

was higher than that of p18-/- alone. In the p21-/- control group, p21-/- HSC progeny were barely

detectable in blood after primary cBMT (Figure 2D) while 63.3% of CD34-LKS cells and 18.9% of

CFCs remained of the p21-/- origin. When normalized to the bone marrow cellularity at the time of

sacrifice, the absolute yield of CD34-LKS cells was in fact significantly lower in the p21-/- transplant

group than in p18-/- recipients.

To further validate the different impacts of p21 and p18 on a per-cell basis, we cultured the CD34-LKS

mixed populations isolated from either the p21-/- or p18-/-p21-/- cBMT group for 14 days and then

checked the ratio of p21-/- or p18-/-p21-/- differentiated cells versus wild-type competitor progeny by the

semi-quantitative PCR. The CD34-LKS cells in both groups were able to differentiate into myeloid

cells during the culture. Consistent with our in vivo findings, the initial higher representation (63.3%)

at HSC level dropped to 25% of HSC progeny in the p21-/- group, whereas the representation

remained (78.9% to 83%) in the p18-/-p21-/- group (Figure 4). Together, these data suggest that a

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progressive decrease of function per HSC is ultimately followed by a possible depletion of the HSC

pool due to the absence of p21, and that such a process can be substantially overcome by deleting

p18 at the stem cell level regardless of p21 absence.

Discussion

Our current study demonstrates that transplanted p18-/- HSCs sustain their self-renewing ability at a

comparable level to unmanipulated wild-type HSCs after multiple serial transfers in both competitive

and non-competitive repopulation models. Furthermore, p18 absence is able to significantly

counteract the accelerated exhaustion of hematopoietic repopulation resulting from p21 deficiency.

Therefore, we have defined for the first time that hematopoietic exhaustion in serial transplant models

can be overcome by targeting a specific cell cycle inhibitor and different cell cycle inhibitors can have

opposite impacts on the outcome.

Both extrinsic and intrinsic factors may account for HSC exhaustion in transplant recipients.

Regarding the extrinsic factors, at least two considerations have been given from previous studies by

others. One is the failure to achieve a physiological concentration of HSCs due to insufficient

mitogens for their regeneration upon the dilution of original donor HSCs in new recipients.2 Another

one is the physical disruption due to transplantation procedure or/and radiation damage (if TBI is used)

which can result in homing defect25 or permanent loss of self-renewal potential6,7,26 of the transplanted

HSCs. However, it is unclear how these extrinsic effects are transmitted onto the intrinsic properties

of transplanted HSCs. In fact, the intrinsic mechanism may also affect HSC exhaustion in an

autonomous manner. Because serial bone marrow transfer usually spans the lifetime of a mouse or

longer beside its imposed transplantation stress, the resultant stem cell exhaustion has been thought

to be attributable to the replicative senescence of the transplanted HSCs, resembling aging of HSC.11

This link is supported by the similarity of limited HSC self-replication in both serial transplant

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recipients and aged mice.27,28 While the aging effect might be cooperative with the extrinsic factors in

leading to hematopoietic exhaustion, the hematopoietic exhaustion was distinguishable from the aged

phenotype to a certain extent in the absence of p18. On one hand, we observed the disappearance

of hematopoietic exhaustion in p18-/- groups as compared to the wild-type cells after serial

transplantation over 3 years, while on the other hand we observed a favored myeloid differentiation of

HSCs that has been similarly observed in old mice and is now considered as a sign for HSC aging.22-

24 Notably, however, the LTRA after each cBMT decreased as compared to the previous one and this

reduction coincided with a relative increase in HSC frequency as defined by the immunophenotype

(Table 1). We observed a dominant p18-/- phenotype in the HSC compartment, but the representation

of p18-/- phenotype in blood did not correlate with the ratio in the stem cell pool after tertiary cBMT.

The reverse relationship was more overt in the p18-/-p21-/- group and the most dramatic in the p21-/-

group (Table 1 and Figure 2D). This suggests a decline in repopulation potential per HSC rather than

an actual loss of the phenotypic HSCs after transplantation. This observation is consistent with

previous studies in both transplanted and untransplanted aged mice.29,30 The similarity between

HSCs in transplant recipients and aged mice suggests that extensive cell divisions may pose

replicative stress on HSCs and ultimately lead to premature cellular senescence. Interestingly, p18

absence is able to significantly counteract the process through a distinct mechanism, yet to be

defined.

The molecular mechanisms that underlie HSC exhaustion are poorly understood. Given that

replicative cellular senescence is likely involved, telomere length was hypothesized to be a critical

cellular timer in mediating the senescent outcome. This hypothesis was supported by the observation

of shortened telomere length after serial transplantation31 and early exhaustion of transplanted HSCs

deficient in telomerase.32 However, overexpression of telomerase in HSCs was not sufficient to

overcome hematopoietic exhaustion, although it could prevent telomere shortening during serial

transplantation.33 In our study, we did not observe a difference in telomere length between p18+/+ and

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p18-/- sBMT groups (data not shown). Therefore, these studies suggest that telomere erosion is not a

sole or indispensable mechanism for stem cell exhaustion.

The p53 and Rb pathways are considered to be the two major activating mechanisms for cellular

senescence in many other cell types.34 p21 is a primary transcriptional target of p53 in mediating cell

cycle arrest, and p16 is a key INK4 protein inhibiting the activity of CDK4/6 in the Rb pathway. Both

cell cycle inhibitors were recently indicated to be involved in radiation-induced HSC senescence.35

However, because p21 is expressed at a higher level in quiescent HSCs,36,37 it is unlikely to play an

indispensable role in HSC senescence. Instead, the Rb pathway might be a more dominant

mechanism for HSC senescence but not HSC quiescence as p16 and its alternative reading frame,

p19ARF were up-regulated in the HSCs from serially transplanted mice (unpublished data in our

laboratory). We therefore hypothesize that under the replicative stress caused by transplantation,

HSCs may undergo senescence, and such a process is accelerated in the absence of p21 due to a

higher cycling status of HSC. In fact, given the increased number of HSCs and normal hematopoietic

potential in p21-/- under homeostatic conditions10 and the unperturbed function of HSCs after ex vivo

targeting of p2138,39, we suggest that the senesent state of p21-/- HSCs in the serial recipients reflect

a secondary effect of p21 absence on HSCs under the repetitive proliferative stress. In the absence

of p18, the senescent outcome can be substantially overcome or delayed due to the antagonistic

effect of p18 absence on the cellular senescence. But it remains to be determined with regard to how

the effect of p18 deletion is mediated by other molecules to compensate the consequence of p21 loss

on hematopoietic exhaustion and more importantly, how p18 interacts with p21 physiologically in

HSCs. Nevertheless, our current study sets the stage regarding our current understanding of the

distinct roles that p18 and p21 play in stem cell regulation. An understanding of the different impacts

that these two CKIs have in HSC kinetics offers molecular targets for possible pharmacological

intervention to improve stem cell functioning in transplant or elderly patients.

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Acknowledgments

This work was supported by NIH grants (DK02761, HL70561) and the Scholar Award from American

Society of Hematology (2003). We specially thank Drs David Franklin and Andrew Stewart for

providing the knockout mouse strains.

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4. Harrison DE, Astle CM, Delaittre JA. Loss of proliferative capacity in immunohemopoietic stem cells caused by serial transplantation rather than aging. J Exp Med. 1978;147:1526-1531

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6. Harrison DE, Astle CM. Loss of stem cell repopulating ability upon transplantation. Effects of donor age, cell number, and transplantation procedure. J Exp Med. 1982;156:1767-1779

7. Mauch P, Rosenblatt M, Hellman S. Permanent loss in stem cell self renewal capacity following stress to the marrow. Blood. 1988;72:1193-1196

8. Harrison DE, Stone M, Astle CM. Effects of transplantation on the primitive immunohematopoietic stem cell. J Exp Med. 1990;172:431-437

9. Pawliuk R, Eaves C, Humphries RK. Evidence of both ontogeny and transplant dose-regulated expansion of hematopoietic stem cells in vivo. Blood. 1996;88:2852-2858

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11. Bell DR, Van Zant G. Stem cells, aging, and cancer: inevitabilities and outcomes. Oncogene. 2004;23:7290-7296

12. Lapidot T. Mechanism of human stem cell migration and repopulation of NOD/SCID and B2mnull NOD/SCID mice. The role of SDF-1/CXCR4 interactions. Ann N Y Acad Sci. 2001;938:83-95

13. Christopherson KW, 2nd, Hangoc G, Mantel CR, Broxmeyer HE. Modulation of hematopoietic stem cell homing and engraftment by CD26. Science. 2004;305:1000-1003

14. Calvi LM, Adams GB, Weibrecht KW, Weber JM, Olson DP, Knight MC, Martin RP, Schipani E, Divieti P, Bringhurst FR, Milner LA, Kronenberg HM, Scadden DT. Osteoblastic cells regulate the haematopoietic stem cell niche. Nature. 2003;425:841-846

15. Cheng T, Scadden DT. Cell cycle entry of hematopoietic stem and progenitor cells controlled by distinct cyclin-dependent kinase inhibitors. Int J Hematol. 2002;75:460-465

16. Yuan Y, Shen H, Franklin DS, Scadden DT, Cheng T. In vivo self-renewing divisions of haematopoietic stem cells are increased in the absence of the early G1-phase inhibitor, p18INK4C. Nat Cell Biol. 2004;6:436-442

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20. Harrison DE, Astle CM, Lerner C. Number and continuous proliferative pattern of transplanted primitive immunohematopoietic stem cells. Proc Natl Acad Sci U S A. 1988;85:822-826

21. Franklin DS, Godfrey VL, O'Brien DA, Deng C, Xiong Y. Functional collaboration between different cyclin-dependent kinase inhibitors suppresses tumor growth with distinct tissue specificity. Mol Cell Biol. 2000;20:6147-6158

22. Sudo K, Ema H, Morita Y, Nakauchi H. Age-associated characteristics of murine hematopoietic stem cells. J Exp Med. 2000;192:1273-1280

23. Geiger H, Rennebeck G, Van Zant G. Regulation of hematopoietic stem cell aging in vivo by a distinct genetic element. Proc Natl Acad Sci U S A. 2005;102:5102-5107

24. Rossi DJ, Bryder D, Zahn JM, Ahlenius H, Sonu R, Wagers AJ, Weissman IL. Cell intrinsic alterations underlie hematopoietic stem cell aging. Proc Natl Acad Sci U S A. 2005;102:9194-9199

25. Plett PA, Frankovitz SM, Orschell-Traycoff CM. In vivo trafficking, cell cycle activity, and engraftment potential of phenotypically defined primitive hematopoietic cells after transplantation into irradiated or nonirradiated recipients. Blood. 2002;100:3545-3552

26. Ross EA, Anderson N, Micklem HS. Serial depletion and regeneration of the murine hematopoietic system. Implications for hematopoietic organization and the study of cellular aging. J Exp Med. 1982;155:432-444

27. Ogden DA, Mickliem HS. The fate of serially transplanted bone marrow cell populations from young and old donors. Transplantation. 1976;22:287-293

28. Kamminga LM, van Os R, Ausema A, Noach EJ, Weersing E, Dontje B, Vellenga E, de Haan G. Impaired hematopoietic stem cell functioning after serial transplantation and during normal aging. Stem Cells. 2005;23:82-92

29. Spangrude GJ, Brooks DM, Tumas DB. Long-term repopulation of irradiated mice with limiting numbers of purified hematopoietic stem cells: in vivo expansion of stem cell phenotype but not function. Blood. 1995;85:1006-1016

30. Morrison SJ, Wandycz AM, Akashi K, Globerson A, Weissman IL. The aging of hematopoietic stem cells [see comments]. Nat Med. 1996;2:1011-1016

31. Allsopp RC, Cheshier S, Weissman IL. Telomere shortening accompanies increased cell cycle activity during serial transplantation of hematopoietic stem cells. J Exp Med. 2001;193:917-924

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32. Allsopp RC, Morin GB, DePinho R, Harley CB, Weissman IL. Telomerase is required to slow telomere shortening and extend replicative lifespan of HSCs during serial transplantation. Blood. 2003;102:517-520

33. Allsopp RC, Morin GB, Horner JW, DePinho R, Harley CB, Weissman IL. Effect of TERT over-expression on the long-term transplantation capacity of hematopoietic stem cells. Nat Med. 2003;9:369-371

34. Ben-Porath I, Weinberg RA. The signals and pathways activating cellular senescence. Int J Biochem Cell Biol. 2005;37:961-976

35. Wang Y, Schulte BA, Larue AC, Ogawa M, Zhou D. Total body irradiation selectively induces murine hematopoietic stem cell senescence. Blood. 2005

36. Cheng T, Shen H, Rodrigues N, Stier S, Scadden DT. Transforming growth factor beta 1 mediates cell-cycle arrest of primitive hematopoietic cells independent of p21(Cip1/Waf1) or p27(Kip1). Blood. 2001;98:3643-3649

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Legends

Figure 1. Continued presence of p18-/- HSC clones without apparent exhaustion during sBMT.

BMNCs were collected from p18-/- or p18+/+ male mice at 2 months of age and transplanted into

lethally irradiated (10 Gy) female recipients (2 x 106 cells/recipient). After long-term engraftment was

established in the primary recipients, the same dose of BMNCs were pooled from the primary

recipients and re-transplanted into lethally irradiated female recipients. The same procedure was

repeated for additional 3 times (A). “0o, I o, II o, III o and IVo” refer to untransplanted, 1st, 2nd, 3rd and

4th sBMT, respectively. “M” and “F” in parentheses indicate male and female respectively. Numbers

under the arrows indicate the months after each sBMT, and numbers in the parentheses are the

cellular age of original transplanted HSCs. In vitro hematopoietic activity was assayed with the

cobble-stone area forming assay (CAFC) and colony forming assay (CFC) as previously described.10

The frequencies of day 35 CAFC (left) and CFC (right) normalized to the untransplanted wild-type

control after different sBMTs are shown in the graphs (B). “*” indicates p < 0.05 (student’s t-test, n=3).

Open and filled bars indicate p18+/+ and p18-/- groups respectively. To further assess the exhaustion

of hematopoietic regeneration, peripheral blood was collected from the recipients 12 months after IVo

sBMT and the PCR-based semi-quantitative analysis for mouse Y chromosome-specific sequence

(Sry) normalized to the housekeeping gene myogenin (Myo) was applied to calculate the contribution

of the original male donor cells (C). The spleen or blood nucleated cells from male and female mice

were mixed at different ratios to acquire the standardization for the semi-quantitative analysis.

According to the standardization generated simultaneously as shown in the bottom panel, the

normalized percentages of donor cells in the blood are indicated under the PCR images in the top

and middle panels. Numbers above the gel images indicate individual p18-/- or p18+/+ transplant

recipients, and engraftment level (%) in each recipient is indicated under the gel image.

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Figure 2. Sustained competitiveness of p18-/- HSCs after multiple rounds of cBMT. BMNCs

from p18-/- or p18-/-p21-/- knockout mice were mixed with an equal number (2x106/each) of competitor

cells (BMNCs from wild-type mice of 2 months old) and injected into lethally irradiated (10 Gy)

recipient mice. 10 months after primary cBMT, 2 x 106 BMNCs from the recipients were re-challenged

with an equal amount of the competitor cells and secondarily transplanted into lethally irradiated

recipients. This procedure was sequentially repeated twice (donor cells were pooled from 3-5

animals and there were 10 recipients/each group for each cBMT). The actual interval between each

cBMT is shown in the illustration (A). Some mice were kept longer than the interval “I o, II o and III o”

refer to 1st, 2nd and 3rd cBMT respectively. “1:1” means that an equal amount of test cells and

competitor cells were co-transplanted into the recipients. “M” and “F” in parentheses indicate male

and female respectively. Numbers under the arrows indicate the months after each cBMT, and

numbers in the parentheses are the cellular age of original transplanted HSCs. Peripheral blood was

collected from the recipients after long-term engraftment during each interval between sequential

cBMTs, and PCR-based semi-quantitative analysis as described in our previous study16 was

performed to determine the contribution of p18-/- or p18-/-p21-/- to the overall hematopoietic

reconstitution. The spleen or blood nucleated cells from wild-type and mutant mice were mixed at

different ratios to obtain a standardization curve for the semi-quantitative analysis. The average

levels of engraftment in blood from p18-/- or p18-/-p21-/- origin 12 months after secondary and 6months

after tertiary cBMT are summarized in the graph (B). Open and filled bars indicate p18-/-and p18-/-

p21-/- transplant groups respectively. Each group includes 8 animals and the difference between p18-

/- and p18-/-p21-/- is significant (p < 0.05) based on the student’s t-test. The representative results after

tertiary cBMT are shown (C) along with the standardization generated simultaneously (bottom panel).

Numbers above the gel images indicate individual animals, and the normalized percentages of test

cells of the total in blood are shown below the images. As a control to confirm the accelerated

exhaustion of hematopoietic repopulation in the absence of p21 as reported previously,10 the identical

cBMT procedure (A) was used to test the repopulating ability of BMNCs from p21-/- mice at the age of

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12 months (D). 8-12 months after the 1st cBMT, there was virtually no detectable p21-/- band in blood

after 30 cycles of the PCR, and there were two blood samples at a barely detectable level (1-3%) of

the engraftment after 35 cycles of the PCR as representatively shown here. This experiment was

intended to confirm a previous claim of HSC exhaustion due to p21 absence10. It was not performed

at the same time as other two groups but the competitor cells were from the same type of donor mice

as described in the Methods. Therefore, the results between p21-/- group and other groups though

with imperfection are still comparable since all these different test cells were directly assessed with

the same type of competitor cells in the irradiated hosts. The singular effect of age would not

substantially contribute to the exhaustion based on earlier studies by others showing no disadvantage

of aged bone marrow cells in the competitive repopulation model.4,27,40

Figure 3. Multilineage differentiation that favors myelopoiesis after an extended long-term

engraftment. The recipients were sacrificed after tertiary cBMT and their BMNCs were collected and

stained with lineage markers. Myeloid (Mac-1+), T (CD3+) and B (B220+) cell lineages were sorted for

semi-quantitative PCR analysis as described in Figure 2C. The actual results are shown in “A”.

Numbers above the gel images indicate individual animals, and the calculated percentages of donor

cells are indicated below the PCR images. The graph in “B” summarizes the contribution of p18-/- or

p18-/-p21-/- donor cells to different lineages in tertiary recipients (6 months after transplantation). Open

and filled bars indicate p18-/-and p18-/-p21-/- transplant groups respectively. “*” indicates p < 0.05 (n=3,

student’s t test).

Figure 4. In vitro growth of LKS cells. CD34-LKS cells were isolated from the recipients of p21-/-

primary cBMT (n=3) or quaternary p18-/-p21-/- cBMT (n=4). 100 CD34-LKS cells were sorted into 96-

well plates and cultured in the Iscove’s MDM supplemented with 100ng/ml SCF, 50ng/ml Flt-3 and

25ng/ml TPO. Cell numbers were counted at 3 time points during the culture. The growth curves

were plotted based on the Mean ± SD of triplicate cultures. An aliquot of the differentiated cells were

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lysed for the semi-quantitative PCR analysis as described in Figure 2. The inserts in the graph are

the PCR results showing the altered genotypic representation (knockout vs. wild-type). Based on the

single cell analysis (Table 1), the initial ratios of p21-/- and p18-/-p21-/- in the starting CD34-LKS cells

were 63.3 % and 78.9% respectively. No differentiation block was observed in both groups based on

the cell morphology under a microscope.

Table 1. Representation of Knockout (KO) in Stem/Progenitor Cell Pools

Clonal analysis of stem/progenitor cells cBMT*

Months post-cBMT

Clonal culture

Mice analyzed

Colonies assayed KO%

Absolute yield of KO CD34-LKS

cells§ IIº 12 CD34-LKS 3 109 92.7 ND$ p18-/-# IIIº 8 CD34-LKS 3 93 76.3 3633 ± 356

CD34-LKS 3 106 99.1 IIº 16 CFC 3 88 97.7 CD34-LKS 2 91 90.1 IIIº 6 CFC 2 88 87.6 CD34-LKS 4 76 78.9

p18-/-

p21-/-

IVº 10 CFC 4 37 40.5

ND$

CD34-LKS 3 60 63.3 p21-/-¥ Iº 8 CFC 3 67 18.9

1452 ± 394

* The cBMT procedure was detailed in our previous publication16 and is further illustrated in Figure 2A. Briefly, test cells (2 x 106 BMNCs/host) from p18-/- or p18-/-p21-/- male mice and an equal number of wild-type BMNCs (competitor cells) from male mice (wild-type litter mates in primary cBMT and then wild-type C57BL/6129/SvF1 mice of 2 months old in subsequent rounds of cBMT) were co-transplanted into lethally-irradiated (10 Gy) female recipients. Overall engraftment levels in blood are presented in Figure 2C-D.

# The representations of p18-/- genotype after after IIº cBMT have been reported in our previous publication.16 All the mice after IVo cBMT in p18-/- group developed T-cell leukemia. Characterization of the leukemic cells and relation to HSC regeneration involving a range of analyses are to be reported in a different manuscript (Yuan Y, et al., submitted).

¥ This experiment was done much later than other two experiments (p18-/ and p18-/-p21-/-) to confirm the accelerated hematopoietic exhaustion due to p21 absence10 as rationalized in the legend of Figure 2D. The competitor cells in all these experiments were from the same type of unmanipulated bone marrow.

§ Absolute yield of KO CD34-LSK cells was calculated according to the bone marrow cellularity per harvest (including ilia, femurs and tibias, n=8), the frequency (by Flow Cytometry) of viable CD34-LKS (being able to respond to growth stimuli in vitro) and KO% in CD34-LKS cells. p<0.01 (t-test, between p18-/- and p21-/- cBMT groups). The lower yield of CD34-LKS cells due to the absence of p21 must have been substantially underestimated since the p18-/- group had undergone two additional rounds of cBMT.

$ ND: not done.

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Figure 1A,B

A

p18-/- orp18+/+(M)

2 X 106

0(2)

I0

(F)

2 X 106

II0

3(5)

2 X 106

III0

(F) 4(9)

(F)

2 X 106

3(12) (F)

IV0

12(24)

Endpoint

Yu H et al

B

0

0.5

1

1.5

2

0

0.5

1

1.5*

* * *

0 o II o I o II o III o

Figure 1C Yu H et al

C

p18+/+SryMyo

Individual mouse 1 2 3 4 5 6

Engraftment(%) 0 66 60 0 0 0

p18-/-SryMyo

Individual mouse 1 2 3 4 5 6 7

Engraftment(%) 65 73 60 66 59 71 71

SryMyo

Cell ratio

0/10

0

10/9

0

25/7

5

50/5

0

75/2

5

90/1

0

100/

0

0

0.4

0.8

1.2

0 0.3 0.6 0.9

y=1.28x-0.14

Mal

e in

the

mix

ed

Popu

latio

m(%

)

Sry/Myo relative intensity

Standardization

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Figure 2AYu H et al

A

2 X 106

2 X 106

0(6)

I0

II0

III02 X 106

2 X 106

2 X 106

2 X 106

2 X 106

2 X 106

1:1

1:1

1:1

1:1

IV0

p18-/- orp18-/-p21-/-

(M)

WT(M)

WT

WT

WT

WT

WT

WT

WT

(M)

(M)

(M)

(F)

(F)

(F)

(F)10

14

8 10

(16)

(30)

(38) (48)

Endpoint

Figure 2B

B

88.50

54.8851.08

34.11

0

20

40

60

80

100

IIO IIIO

Perc

enta

ge in

Tot

al

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Figure 2C Yu H et al

C

p18-/-p18+/+

Individual mouse 1 2 3 4 5 6 7 8

Engraftment(%) 58 86 57 49 38 57 39 54p18-/-

Individual mouse 1 2 3 4 5 6 7 8p18+/+p18-/-

Engraftment(%) 17 36 62 40 13 23 41 39

Cell ratio

0/10

0

10/9

0

25/7

5

50/5

0

75/2

5

90/1

0

100/

0

p18-/-p21-/-

p18+/+

p18-/- 0

0.4

0.8

1.2

0 0.4 0.8 1.2

y=1.00+0.07

Relative p18-/- signal in totalp18-

/-in

the

mix

ed

Popu

latio

n(%

)

Standardization

Figure 2DYu H et al

D

Individual mouse 1 2 3p21+/+p21-/-

Engraftment(%) 1 0 3p21-/-

1/99

2/98

5/95

10/9

0

Cell ratiop21+/+

P21-/-Standardization

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Figure 4Yu H et al

p18+/+

p18-/- (83%)

p21+/+

p21-/- (25%)

Days in culture

Cel

l num

ber (

X104

)

0.01

2

4

6

Figure 3 Yu H et al

AIndividual mouse 1 2 3 1 2 3 1 2 3

p18+/+

P18-/-

Engraftment(%) 100 85 77 76 62 64 69 53 47

G/M T B

Individual mouse 1 2 3 1 2 3 1 2 3p18+/+

p18-/-

Engraftment(%) 90 73 74 35 37 26 76 63 49

G/M T B

p18-/-

p18-/-p21-/-

0

20

40

60

80

100

*

G/M T B

Engr

aftm

ent(

%)B

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