Therapeutic targeting of a stem cell nicheGregor B Adams1,4, Roderick P Martin1,4, Ian R Alley1,4, Karissa T Chabner1,4, Kenneth S Cohen1,4,Laura M Calvi3, Henry M Kronenberg2 & David T Scadden1,4

The specialized microenvironment or niche where stem cells

reside provides regulatory input governing stem cell function.

We tested the hypothesis that targeting the niche might

improve stem cell–based therapies using three mouse models

that are relevant to clinical uses of hematopoietic stem (HS)

cells. We and others previously identified the osteoblast as a

component of the adult HS cell niche and established that

activation of the parathyroid hormone (PTH) receptor on

osteoblasts increases stem cell number1–3. Here we show that

pharmacologic use of PTH increases the number of HS cells

mobilized into the peripheral blood for stem cell harvests,

protects stem cells from repeated exposure to cytotoxic

chemotherapy and expands stem cells in transplant recipients.

These data provide evidence that the niche may be an

attractive target for drug-based stem cell therapeutics.

As a source of cells for replenishing hematopoietic tissue, adult HScells provide a lifesaving treatment for many hematological disorders.However, low numbers of HS cells can be limiting in the settings ofboth autologous and allogeneic transplants. Individuals with Hodgkinor non-Hodgkin lymphoma may require intensive salvage chemo-therapy and stem cell rescue after multiple rounds of cytotoxic agents.It is estimated that 10–20% of such individuals may fail to mobilizesufficient HS cells to safely accomplish this procedure4. In addition,o50% of individuals requiring an allogeneic HS cell transplant maybe able to find a suitable matched donor5. Umbilical cord blood mayoffer a valuable resource for such patients, who are often members ofethnic and racial groups typically underrepresented in unrelateddonor banks6,7. However, individual units of cord blood contain toofew HS cells to allow them to be transplanted into adults weighing440 kg without a substantial delay in engraftment. Therefore,methods that increase the number of stem cells in HS cell harvestsor increase the efficiency of engraftment of small numbers of stem cellscould provide substantial clinical benefit. Strategies to accomplishthese goals have not been successfully developed to date.

One underexplored strategy is to target the HS cell niche. Adult HScells reside within the context of a complex microenvironment ofdifferent cell types and extracellular matrix molecules that dictate stemcell self-renewal and progeny production in vivo8. Therefore, thecomponents of this niche may provide targets for therapies aimed at

altering stem cell fate. Within the adult bone marrow, HS cells havebeen shown to reside in proximity to the endosteal surface of boneand blood vessels9–12. This relationship of HS cells to bone suggested apossible role in the HS cell niche for osteoblasts13,14. Osteoblasts areindeed a key component of the adult HS cell niche in vivo1–3. Ourprevious work used a transgenic mouse model in which a constitu-tively active form of the PTH/PTH-related peptide (rP) receptor wasexpressed specifically in cells of the osteoblastic lineage. This led to aspecific expansion of the HS cell pool mediated by activation ofthe Notch pathway. We further demonstrated that these effectscould be recapitulated through administration of exogenous PTH toa wild-type mouse1.

Here we test the hypothesis that stimulation of the adult HS cellniche, rather than the stem cell itself, can provide therapeutic benefitin animal models that are pertinent to clinical stem cell transplanta-tion15. We addressed this hypothesis in three clinically relevantsituations: resident stem cell expansion, protection of resident stemcells during myelotoxic injury and engraftment of exogenously deliv-ered stem cells to the niche. Specifically, we examined whether PTHtreatment could improve outcomes in the following in vivo models:(i) granulocyte-colony stimulating factor (G-CSF) mobilization of HScells in normal donors, (ii) preservation of stem cells for transplantafter multiple rounds of cytotoxic chemotherapy and (iii) hemato-poietic reconstitution after myeloablation and stem cell rescue.

First, we documented an absence of PTH/PTHrP receptor messagein HS cells to be sure that effects of PTH were mediated bymicroenvironmental cells (Supplementary Fig. 1 online). Next, weexamined whether PTH could increase HS cells capable of beingmobilized into the circulation by G-CSF. C57Bl/6 mice were treatedwith PTH or mock treated with vehicle alone for 5 weeks. At the endof the treatment period, the mice underwent a standard mobilizationprocedure with G-CSF for 5 d (Fig. 1a). The treatment with PTH didnot result in any significant alteration of complete blood counts(CBCs), including white blood count (WBC; Fig. 1b), neutrophils,hemoglobin or platelets (Supplementary Fig. 2a–c online). Mobiliza-tion with G-CSF led to an increase in WBC and neutrophils, whichwere not altered by the concurrent treatment with PTH (Fig. 1b andSupplementary Fig. 2a). After mobilization with G-CSF, the numberof colony-forming units culture (CFU-Cs) mobilized into the peri-pheral circulation was significantly (P o 0.029) increased compared

Received 28 April 2006; accepted 1 December 2006; published online 21 January 2007; doi:10.1038/nbt1281

1Center for Regenerative Medicine, Massachusetts General Hospital, Harvard Medical School, 185 Cambridge Street, Boston, Massachusetts 02114, USA.2Endocrine Unit, Massachusetts General Hospital, Harvard Medical School, 55 Fruit Street, Massachusetts 02114, USA. 3Endocrine Unit, Department of Medicine,University of Rochester School of Medicine, 601 Elmwood Avenue, Rochester, New York 14642, USA. 4Harvard Stem Cell Institute, Harvard University, 42 Church Street,Cambridge, Massachusetts 02138, USA. Correspondence should be addressed to D.S. (scadden.david@mgh.harvard.edu).

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with nonmobilized controls. There were no significant differences inCFU-Cs mobilized in the mock-treated or PTH-treated mobilizedmice (Fig. 1c), consistent with previous studies demonstrating thatPTH treatment has no effect on progenitor cells1.

Analysis of the HS cell content of the peripheral blood wasaccomplished by two independent means: immunophenotyping(lin–Sca-1+c-Kit+Flk-2–)16 and evaluation of function as measuredby a competitive repopulation assay (CRA) of irradiated mice17,18.Mobilization with G-CSF increased the immunophenotypic HS cellcontent in the blood (control nonmobilized, 743 ± 36 cells/ml; controlmobilized, 6,189 ± 661 cells/ml; PTH nonmobilized, 758 ± 120 cells/ml;PTH mobilized, 12,340 ± 3,407 cells/ml; Fig. 1d). Similar results wereobtained when the number of HS cells was assessed by CRA (Fig. 1e).These data indicate that PTH treatment does not result in an alterationof mature cell counts or progenitor cell mobilization with G-CSF.However, PTH treatment increases the number of HS cells in the bonemarrow that can be mobilized into the peripheral circulation with astandard mobilization regimen, resulting in an increased number of HScells in the peripheral circulation.

Many individuals receiving multiple rounds of chemotherapy sub-sequently receive autologous HS cell transplants. A sizable proportionof patients receiving chemotherapy also require growth-factor sup-port. However, chemotherapy in combination with G-CSF may lead toa significant depletion (P o) of the bone marrow HS cell pool19–22.Therefore, we tested whether PTH during multiple rounds of

chemotherapy with or without supportive G-CSF therapy leadsto preservation of the HS cell pool. Mice received 5 mg ofcyclophosphamide, a commonly used cancer chemotherapy, onceevery 2 weeks for a total of four cycles (Fig. 2a). One day after eachof the four cyclophosphamide treatments, mice were treated witheither saline, G-CSF for 8 d21,22, PTH for 11 d or a combination of G-CSF and PTH. At the end of the 8-week treatment period, half of themice were killed and the HS cell pool in the bone marrow was assessedby CRA. In the other half, 2 weeks after the end of the treatmentcycles, HS cell mobilization by G-CSF into the blood was assessed byCRA (mimicking the setting of autologous transplantation afterchemotherapy). Throughout the protocol, peripheral blood CBCswere obtained every 2–3 d, but individual mice were only bled weeklyto avoid excess stress.

Pooled CBC data (Fig. 2b and Supplementary Figs. 3a–c) showedthat G-CSF increased WBC and neutrophil counts after chemo-therapy, unaffected by the addition of PTH. Similarly, in mice thatdid not receive G-CSF there were no differences in hematologicalresponse to chemotherapy whether they received PTH or not(although a slight advantage from PTH treatment with G-CSF mayhave emerged after the fourth cycle of chemotherapy). DocumentingHS cell number by flow cytometry after repeated exposure tochemotherapeutic agents is known to be problematic because ofinfidelity of surface markers with this type of stress21,22. Therefore,the HS cell pool could be reliably measured only by functional CRA

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Figure 1 Increased HS cell mobilization into the peripheral circulation after PTH treatment. (a) Diagrammatic representation of the experimental procedure

to analyze the ability of PTH treatment to augment HS cell mobilization to the peripheral circulation following a standard mobilization regimen. (b) WBC

analysis of the peripheral blood of mice treated with PTH or control (saline) for 5 weeks followed by 5 d of G-CSF mobilization or control. Each point

represents the mean of 5 individual mice ± s.e.m. (c) CFU-C analysis of the peripheral blood after mobilization with G-CSF or control (n ¼ 4). Red bars

represent the mean value. (d) Measurement of the HS cell content of the peripheral blood by immunophenotypic analysis at day 40. Representative

flow cytometry plot of five independent experiments of Sca-1 and c-Kit expression of the lin–Flk-2– cells is shown with the mean frequency indicated.

(e) Competitive repopulation assay at 16 weeks of mice treated with PTH or control that underwent mobilization with G-CSF (control treated, n ¼ 5;

PTH treated, n ¼ 4). Ly., lymphoid cells; My., myeloid cells.

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studies. These studies demonstrated that PTH treatment increased theHS cell pool in mice that did not receive G-CSF as supportive therapy(Fig. 2c). In the animals treated with G-CSF alone, there was asignificant depletion (P o 0.029) of the HS cell pool, as has beendescribed by others19,21,22. However, treatment with both G-CSF andPTH led to relative preservation of the HS cell pool (Fig. 2c).

Similarly, analysis of the mobilization of HS cells into the peripheralcirculation showed that, in mice that did not receive supportive G-CSFtherapy during the myeloablative chemotherapy, there was mobiliza-tion of HS cells into the circulation, and mobilization was increasedwith prior PTH treatment (Fig. 2d). However, mice that receivedsupportive growth factor (G-CSF) therapy alone showed little to nomobilization of HS cells into the peripheral circulation. This waspartially reversed by concurrent treatment with PTH (Fig. 2d).

Taken together, these studies show that targeting the niche canprotect, or even expand the resident HS cell pool in the bonemarrow during myelotoxic chemotherapy. The benefit of PTH wasespecially evident when G-CSF supportive therapy was used inconjunction with chemotherapy.

We previously demonstrated that mice that received a transplant oflimiting numbers of bone marrow mononuclear cells had an increasedsurvival advantage if they received daily PTH after transplant com-pared with those treated with a saline control1. Because theseobservations were made over a short time period and were associatedwith marked increases in marrow cellularity, we could not rule out thepossibility that the PTH treatment stimulated the cells to differentiatemore rapidly into mature cells, at the expense of HS cell self-renewal.Therefore, we performed experiments (Fig. 3a) to determine whetherPTH treatment depleted HS cells after transplant. C57Bl/6 (CD45.2)mice were lethally irradiated and injected with 5 � 105 bone marrowmononuclear cells from a B6.SJL (CD45.1) donor. This dose of cellsensured 100% survival of the mice. The day after transplantation, the

mice were treated with PTH or mock treated with vehicle alone. Every2–3 d after transplant, we analyzed CBCs from each group of mice toassess hematopoietic recovery, but again, bled an individual mouseonly once per week to avoid stress. Treatment with PTH had no effecton the recovery of the WBC, neutrophils, hemoglobin or platelets inthe 6-week time period after transplant (Fig. 3b and SupplementaryFig. 4a–c online). Recent reports have indicated that the level ofexpression of Notch ligands influences the fate choice of HS cells,particularly of the B- and T-lymphoid lineages23. Because exposure ofosteoblasts to PTH results in upregulation of Jagged1 (refs. 1,24), wealso assessed the repopulation of the B- and T-lymphoid lineages. Aswith other lineages, no effect was seen in the repopulation of theCD4+, CD8+ or B220+ lymphocytes (Fig. 3c).

Quantification of the number of HS cells in the bone marrow aftertreatment with PTH or saline control by immunophenotypic analysesdemonstrated a significant (P o 0.011) increase in the PTH-treatedgroup (Fig. 3d). To functionally analyze the HS cell content of thebone marrow, we carried out competitive secondary transplants afterthis 6-week engraftment period. The primary recipients were killedand 5 � 105 bone marrow mononuclear cells were mixed with 2.5 �105 bone marrow mononuclear cells from a C57Bl/6 mouse. Thesecells were then injected into a lethally irradiated C57Bl/6 secondaryhost (Fig. 3a). Measuring short-term (6 weeks) and long-term (16weeks) HS cells in the PTH-treated or mock-treated mice, we observedan increase in the engraftment of cells obtained from the PTH-treatedmice at each time point (Fig. 3e,f). Taken together, these results showthat PTH treatment of transplanted recipients resulted in an expan-sion of HS cells that functioned normally over prolonged periods.Stimulation of the HS cell niche did not result in an increase in maturecell production at the expense of stem cell self-renewal; rather, PTHtreatment resulted in the expansion of exogenous stem cells deliveredto the niche.

(Day 71)Assess HSC

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Figure 2 PTH treatment preserves HS cell function during multiple rounds of chemotherapy. (a) Diagrammatic representation of the experimental procedure

to analyze the effects of PTH (80 mg/kg/d) treatment during multiple rounds of chemotherapy with cyclophosphamide (Cytoxan, CTX; 5 mg). G-CSF, 5 mg/d.

BM, bone marrow. PB, peripheral blood (b) WBC analysis of the peripheral blood of mice treated with PTH, G-CSF, PTH plus G-CSF or control (saline) during

the four cycles of chemotherapy. Each point represents the mean of three individual mice ± s.e.m. (c) Measurement of the HS cell content of the bone

marrow immediately after the fourth cycle of chemotherapy by CRA at 16 weeks (n ¼ 4). (d) Measurement of the HS cell content of the peripheral blood

of mice that underwent mobilization with G-CSF by CRA at 16 weeks (n ¼ 4).

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We and others have begun to delineate the cellular and molecularcomponents of the adult HS cell niche1,2,25,26. Some of these studieshave also been able to identify components of the niche that may bemanipulated to alter the survival of mice in response to severemyelotoxic stress1,25,26. We assessed the potential of PTH as a stemcell therapy by directly examining stem cell effects in three clinicallyrelevant models of mobilization, myelotoxic chemotherapy andengraftment after bone marrow transplantation. Pharmacologicmanipulation necessarily provides the potential for other cell typesto be affected. Although secondary effects of PTH on other cellscannot be excluded here, we based our experiments on observationsmade in a genetic model where the PTH receptor was activatedspecifically in osteoblasts and an effect on HS cell number wasobserved. In addition, we demonstrated that one important celltype, the HS cell, did not express the PTH receptor. Yet, in each ofthe three models used, PTH induced a beneficial effect on HS cells,which supports the argument for a niche-based effect.

It remains to be shown that the effects of stimulating the HS cellniche with PTH will be similar in humans. The data presented heresuggest several settings in which it may be reasonable to test thispossibility. First, if the efficiency of stem cell harvesting can beimproved by increasing the number of stem cells that can bemobilized, the time required to undergo leukopheresis for harvestingcould be reduced. This may be particularly attractive in concert withnew agents being developed to increase mobilization efficiency, astrategy likely to act at least additively with the use of PTH to increasethe stem cell pool.

Second, repeated rounds of cytotoxic chemotherapy (particularlywhen combined with hematopoietic growth factors27) impair bonemarrow function, often limiting the ability of patients to receive futurerounds of optimal chemotherapy doses or limiting the ability toobtain suitable stem cell products before a salvage bone marrowtransplant. Strategies to maintain stem cell number and function inthese clinical situations would therefore be desirable. Recent reportshave shown that treatment with G-CSF actually inhibits osteoblastfunction28, which may affect HS cell function in the long term. In themice that received G-CSF, PTH may have counteracted the negativeinfluence of hematopoietic growth factors on cells of the osteoblasticlineage. Additionally, repeated rounds of chemotherapy can damagestem cells further, leading to secondary stem cell diseases such asmyelodysplasia and acute myelogenous leukemia. Although notdirectly demonstrated here, the protection of stem cell number andfunction provided by PTH might influence the relative competitivebalance between normal and genotoxin-induced dysfunctional stemcells that account for the secondary complications of chemotherapy.

Third, the data demonstrate that PTH treatment after transplantleads to increased engraftment of the HS cell compartment throughincreased expansion of the stem cell pool. Taken together withprevious results demonstrating a survival advantage of PTH-treatedanimals transplanted with limiting numbers of stem cells1, theseresults suggest that PTH treatment can increase the efficiency of agiven dose of stem cells. If similar effects can be demonstrated inhumans, it may be possible to use fewer stem cells in transplantationprotocols. This would be particularly useful in the setting of umbilical

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Figure 3 PTH treatment augments HS cell engraftment after bone marrow transplantation. (a) Diagrammatic representation of the experimental procedure to

analyze the ability of PTH treatment to increase HS cell engraftment after bone marrow transplant. BM, bone marrow. (b) WBC analysis of the peripheral

blood of mice treated with PTH or control (saline) during the 6-week treatment period of the primary recipients. Each point represents the mean of six

individual mice ± s.e.m. (c) Lymphocyte reconstitution in mice treated PTH or control during the 6-week treatment period. Each point represents the mean

of three individual mice ± s.e.m. (d) Measurement of the HS cell content of the bone marrow by immunophenotypic analysis. Representative flow cytometry

plot of four independent experiments of Sca-1 and c-Kit expression of the lin–Flk-2– cells. (e,f) Competitive repopulation analysis of the bone marrow

mononuclear cells of the primary recipients measured at 6 weeks (e; n ¼ 4) and 16 weeks (f; n ¼ 5) in the secondary recipients. Ly., lymphoid cells;

My., myeloid cells.

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cord blood transplantation in adults, where low stem cell numberrestricts the utility of this otherwise very valuable stem cell source.

In summary, we provide a proof of principle that targeting the adultHS cell niche is a viable therapeutic option for therapies dependantupon HS cells. Using three clinically relevant model systems, wedemonstrate that we can expand stem cells resident in the niche,protect stem cells resident in the niche during myelotoxic stress andalso increase the ability of exogenously delivered stem cells to engraftin the stem cell niche. These studies provide a basis for clinical trialsaimed at increasing the effectiveness of HS cell therapies throughtargeting of the HS cell niche. Whether this strategy is a reasonablemeans of modifying stem cell responses in other tissues should beexperimentally explored.

METHODSIn vivo treatment. C57Bl/6 andB6.SJL mice (Taconic) were obtained and used

in accordance with the Subcommittee on Research Animal Care of the

Massachusetts General Hospital guidelines. Mice were housed in sterilized

microisolator cages and received autoclaved food and water ad libitum. For

PTH treatment, mice were injected intraperitoneally with rat PTH(1-34)

(Bachem Bioscience) at a dose of 80 mg/kg/day. For both mobilization and

growth factor treatment after chemotherapy, mice were injected intraperitone-

ally with recombinant human G-CSF (Amgen) at a dose of 5 mg/day.

Cyclophosphamide (Bristol-Myers Squibb) was injected intraperitoneally at a

dose of 5 mg per mouse. In all experiments, control mice received intra-

peritoneal injections of saline.

Complete blood count analysis. Peripheral blood was collected from tail bleeds

into Microtainer tubes (Becton Dickinson) containing EDTA. Twenty micro-

liters of blood was analyzed using a Hemavet 850FS Multispecies Hematology

Analyzer (Drew Scientific).

CFU assay. Mononuclear cells isolated from peripheral blood was assessed for

CFU-C frequency by culturing in medium containing methylcellulose (M3434;

Stem Cell Technologies) as described previously29.

Competitive repopulation assay. For qualitative measurement of HS cell

frequency in the peripheral circulation, we collected 300 ml of peripheral blood

from tail bleeds into Microtainer tubes containing lithium heparin. The red

cells were lysed with ACK Lysing Buffer and the mononuclear cells were mixed

with 2.5 � 105 bone marrow mononuclear cells from a B6.SJL mouse. For

measurement of bone marrow HS cell frequency, the mice were killed with CO2

and the bone marrow mononuclear cells were isolated by flushing the bone

marrow cavity with fully supplemented medium. We then mixed 2.5 � 105

bone marrow mononuclear cells with an equal number of bone marrow

mononuclear cells from a wild-type competitor mouse. These cells were then

injected into recipient B6.SJL or C57Bl/6 mice, lethally irradiated 24 h

previously with 10 Gy of radiation. The relative contribution of engraftment

from the different cell sources was assessed by flow cytometry of the peripheral

blood for CD45.1 and CD45.2 antigens in both the myeloid (defined as Side

ScatterhiMac-1+) and lymphoid (defined as Side ScatterloCD3+/B220+) fraction

of cells.

Flow cytometry. Peripheral blood was collected from tail bleeds into Micro-

tainer tubes containing EDTA. The blood was diluted 1:1 with PBS and

incubated with anti-CD45.1, CD45.2, CD3, B220 and Mac-1 antibodies (BD

Biosciences) for competitive repopulation analysis. Quantification of the HS

cell frequency was performed as previously described11, with the addition of an

anti-Flk-2 antibody (BD Biosciences). To analyze lymphocyte engraftment, we

incubated peripheral blood with anti-CD3, CD4, CD8 and B220 antibodies.

After incubation with the antibodies, the samples were fixed and the red cells

lysed using BD fluorescence-activated cell sorting (FACS) lysing solution (BD

Biosciences). The relative contribution of the different cell populations was

assessed by flow cytometry using a FACScalibur cytometer and Cell Quest

software (Becton Dickinson).

Statistical analysis. Data were analyzed using the nonparametric

Mann-Whitney test as appropriate for the data set. P o 0.05 was

considered significant.

Requests for materials. D.T.S. (scadden.david@mgh.harvard.edu).

Note: Supplementary information is available on the Nature Biotechnology website.

ACKNOWLEDGMENTSThe authors would like to thank the Cheryl Chagnon Lymphoma ResearchFund for their generous support. Financial support for this work was alsoprovided by the Burroughs Wellcome Fund, Doris Duke Charitable Trust(D.T.S.) and the National Institutes of Health (G.B.A., H.M.K., D.T.S.).

AUTHOR CONTRIBUTIONSG.B.A., K.S.C., L.M.C., H.M.K. and D.T.S. designed the experiments. G.B.A.,R.P.M., I.R.A. and K.T.C. performed the experiments. G.B.A., R.P.M., I.R.A.,K.T.C., K.S.C., L.M.C., H.M.K. and D.T.S. analyzed the data obtained. G.B.A.and D.T.S. drafted the manuscript. G.B.A., R.P.M., I.R.A., K.T.C., K.S.C., L.M.C.,H.M.K. and D.T.S. provided revisions to the manuscript.

COMPETING INTERESTS STATEMENTThe authors declare competing financial interests (see the Nature Biotechnologywebsite for details).

Published online at http://www.nature.com/naturebiotechnology/

Reprints and permissions information is available online at http://npg.nature.com/

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944 VOLUME 25 NUMBER 8 AUGUST 2007 NATURE BIOTECHNOLOGY

Erratum: PeopleNature Biotechnology 25, 692 (2007); published online 1 June 2007; corrected after print 8 August 2007

In the version of this article initially published, Anne Wojcicki was incorrectly referred to as Dr. Wojcicki rather than Ms. Wojcicki.

Corrigendum: Therapeutic targeting of a stem cell nicheGregor B Adams, Roderick P Martin, Ian R Alley, Karissa T Chabner, Kenneth S Cohen, Laura M Calvi, Henry M Kronenberg & David T ScaddenNature Biotechnology 25, 238–243 (2007); published online 21 January 2007; corrected after print 8 August 2007

In the version of this article initially published online 21 January 2007, the y-axes of Figures 1e, 2c and 2d are labeled incorrectly. They should be labeled “% CD45.1 in PB.” The error has been corrected for all versions of the article.

ERRATA AND CORR IGENDA©

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