Proc. Nati. Acad. Sci. USAVol. 82, pp. 2508-2512, April 1985Medical Sciences

Bone marrow engraftment efficiency is enhanced by competitiveinhibition of the hepatic asialoglycoprotein receptor

(bone marrow transplantation/splenic colony-forming unit/liver sequestration/peanut agglutinin)

WOLFRAM E. SAMLOWSKI*t AND RAYMOND A. DAYNEStDepartments of *Internal Medicine (Hematology/Oncology) and of tPathology, University of Utah School of Medicine, Salt Lake City, UT 84132

Communicated by M. M. Wintrobe, December 17, 1984

ABSTRACT The efficiency of pluripotent stem cell en-graftment following bone marrow transplantation is predicat-ed upon many poorly understood factors. These include theprocesses by which intravenously injected stem cells circulateand localize in microenvironments that contain the stromal el-ements necessary to facilitate their continued proliferation.We have recently established that lymphoid cells that bind thelectin peanut agglutinin are subject to prolonged sequestrationfollowing their interaction with the hepatic asialoglycoproteinreceptor. Since bone marrow stem cells are also known to bindpeanut agglutinin, we hypothesized that the physiologic func-tion of the asialoglycoprotein receptor might significantly im-pair their ability to localize in anatomic sites where they areable to proliferate. Competitive inhibition with asialoglycopro-teins was employed to establish a temporary receptor blockadeduring the initial 3-4 hr after transplantation. This procedureresulted in a 5- to 10-fold increase in splenic hematopoieticcolony formation. Our findings suggest that inhibition of theliver asialoglycoprotein receptor during murine bone marrowtransplantation results in more efficient stem cell localizationto hematopoietic-inducing microenvironments. This enhance-ment in engraftment efficiency was paralleled by a more rapidrecovery of peripheral blood leukocyte and platelet counts, anincrease in megakaryocytic colony formation, as well as in-creased recipient survival. Techniques designed to inhibit theliver sequestration of bone marrow stem cells may have directapplicability to human bone marrow transplantation proce-dures.

Bone marrow transplantation has gained widespread accept-ance in the treatment of numerous hematologic and neoplas-tic disorders, although many crucial aspects associated withthis procedure remain poorly understood. One of these as-pects relates to the process by which intravenously injectedstem cells circulate and arrest in specific anatomic compart-ments (such as spleen and bone marrow cavity), which con-tain the facilitative stromal elements necessary for effectiveproliferation and hematopoiesis (1). We have recently identi-fied the existence of an interaction between lymphoid cellsthat bind the lectin peanut agglutinin (PNA) and the hepaticasialoglycoprotein receptor (2). This receptor has been pos-tulated to function in the removal of galactose-terminal gly-coproteins from the circulation (3). Our studies indicatedthat this hepatic receptor can additionally bind to circulatinglymphoid cells, resulting in a sequestration of intravenouslyinjected PNAhi cells within the liver. This sequestrationcauses a decrease in the percentage of injected cells that arecapable of entering usual sites of localization. The liver se-questration of PNAhi cells, such as cortical thymocytes andneuraminidase-treated T cells, can be competitively inhibit-ed by the coinjection of asialoglycoproteins, thus enabling

the infused cells to localize within appropriate secondarylymphoid organs (2).The pluripotent stem cell-containing fraction of the bone

marrow is known to be agglutinated by the lectin PNA (4).These cells therefore possess a surface phenotype that islikely to result in their liver sequestration following intrave-nous infusion (2). We have hypothesized that a physiologicfunction of the liver may significantly impair the efficiencyof successful bone marrow transplantation, due to its abilityto specifically sequester pluripotent stem cells. Since theadult liver does not appear to support hematopoiesis (5), aprocedure designed to temporarily block the uptake of stemcells by this organ may prove beneficial to the engraftmentprocess.The objective of this investigation was to establish wheth-

er the liver-associated asialoglycoprotein receptor has a neg-ative impact on the efficiency of bone marrow stem cell en-graftment. Further, a simple procedure is described, whichprovides a means to enhance (5- to 10-fold) the colony-form-ing potential of transfused syngeneic bone marrow. The po-tential for clinical application of these observations is dis-cussed.

MATERIALS AND METHODS

Animals. Six- to 10-week-old C3H/HeN mice were ob-tained from the animal production facility of the NationalCancer Institute (Bethesda, MD) or bred in our own colony.All mice were housed at a maximal density of six animals per18 x 28 cm cage and maintained on Wayne sterilizable LabBlox (Wayne Pet Food, Chicago) and acidified water ad lib.Mice were then age and sex matched at the onset of eachexperiment.

Preparation of the Bone Marrow Cells. Mice were sacri-ficed by cervical dislocation and their intact femurs weresurgically excised. The proximal femoral head was removedwith a scalpel and the marrow was aseptically irrigated fromthe central cavity with RPMI 1640 medium (GIBCO) supple-mented with 10% fetal calf serum (complete medium) (SterileSystems, Logan, UT). The marrow stroma was gently disso-ciated with sterile forceps and pipetting. Erythrocytes in thecell suspension were lysed with sterile buffered isotonic am-monium chloride. The nucleated marrow cell suspensionswere always .98% viable by trypan blue exclusion.

Radiolabeling of Cells. Bone marrow cells were radiola-beled with 51Cr (as sodium chromate, specific activity = 50-400 mCi/mg of Cr; 1 Ci = 37 GBq; Amersham). Cells wereadjusted to 108 viable cells{er ml and incubated for 1/2 hr at37°C with 50-100 ,Ci of s Cr. The radiolabeled cells werethen washed three times in complete medium and adjusted to107 viable cells per ml in complete medium, and 0.2 ml of the

Abbreviations: ASF, asialofetuin; CFU-S, splenic colony-formingunit; PNA, peanut agglutinin.

2508

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Dow

nloa

ded

by g

uest

on

Aug

ust 2

1, 2

020

Proc. Natl. Acad. Sci. USA 82 (1985) 2509

cell suspension was injected intravenously into normal syn-geneic recipient mice.

Isotope Quantitation. Recipient animals were sacrificed atvarious times after being infused with radiolabeled bonemarrow cells. Peripheral blood (0.2 ml) was sampled at eachtime point to ascertain the fraction of the injected cells re-tained within the circulation via a Beckman gamma 8000counter.Bone Marrow Transplantation. Prior to transplantation,

the experimental animals were pretreated for 2 days with 0.5mg of gentamycin sulfate given intraperitoneally. Through-out the course of the experiment, recipient mice were givenoral nonabsorbable antibiotics (35 mg of neomycin and 2500units of polymyxin B per 200 ml of drinking water). This pre-treatment was employed to reduce mortality from infectionduring the engraftment process. Recipient mice were lethallyirradiated with 8.12-9.28 Gy per whole body at 5.80 Gy/minby utilizing a Gammator (Isomedix, Parsippany, NJ) con-taining a cesium source. Within 1 hr after irradiation, theanimals were infused with the specified numbers of nucleat-ed marrow cells with or without the appropriate competitiveinhibitor of the liver asialoglycoprotein receptor. The en-grafted animals were subsequently placed into sterile cageswith filter bonnets and given sterile food and antibiotic-con-taining water ad lib.

Assay for Spleen Colony Formation. The experimental ani-mals were sacrificed on day 10 or 11 after engraftment, andtheir spleens were removed and fixed in 10% buffered for-malin for 24 hr. Spleen colonies were enumerated as de-scribed by Till and McCulloch (6). In addition, formalin-fixed specimens of femoral marrow and splenic tissue takenfrom the experimental groups were embedded and sectionedfor histologic evaluation.

Competitive Inhibitors of Liver Uptake. Asialofetuin (ASF)was prepared by mild acid hydrolysis (0.1 M HCl at 80'C for30 min) of fetuin type III (Sigma). The extent of desialationachieved was determined by measuring the release of freesialic acid from the glycoprotein by the thiobarbiturate assay(7). The desialated fetuin was then dialyzed exhaustivelyagainst phosphate-buffered saline (pH 7.40) and stored fro-zen. Protein content was determined by the Lowry assay.

Statistical Analysis. Comparisons of the experimentalgroups were made by utilizing the standard two-tailed t test.

RESULTSSince normal T lymphocytes are known to rapidly leave thecirculation and localize in tissue sites following intravenousinfusion (2, 8), our first experiment was designed to deter-mine whether nucleated bone marrow cells behaved in a sim-ilar manner. Bone marrow cells were radiolabeled with 51Crand injected into normal syngeneic mice. Samples of periph-eral blood were collected at varying times (10 min to 3 hrafter injection) to determine the percentage of the infusedcells that remained in the circulation. This experiment (Fig.1) established that a very rapid rate of tissue localization ofthe infused bone marrow cells takes place, so that by 1 hr,>97% of the cells had been cleared from the bloodstream.The majority of the radiolabeled bone marrow cells was

found to localize in the liver, spleen, lungs, and bone marrowcompartment after leaving the bloodstream (data notshown). This experiment suggested that the initial tissue lo-calization of bone marrow cells might well determine wheth-er pluripotent stem cells would be able to reach a hematopoi-etic-inducing microenvironment. Since 6-19% of nucleatedbone marrow cells are known to be PNAhi (9), we hypothe-sized that a potential benefit to bone marrow engraftmentmight be derived from blocking the asialoglycoprotein recep-tor-mediated uptake of PNAh, stem cells by the liver duringthe time when the initial localization process takes place. Todetect changes in the localization of the pluripotent stem cell

100

E

t.-O

80

60-

40-

20

1Time. hr

3

FIG. 1. Clearance of nucleated bone marrow cells from the pe-ripheral blood. Nucleated bone marrow cells were radiolabeled with51Cr and injected into normal 8-week-old C3H/HeN mice. At vary-ing times after injection, 0.2 ml of blood was assayed for the percent-age of radiolabeled cells still in the circulation. Assuming that 0.2 mlof blood represents -10% of the blood volume of a 25-g mouse, theclearance of nucleated marrow cells from the entire circulatingblood pool was calculated. The SEM was always <1% of the inject-ed counts.

population, which represents only about 0.1% of the totalnucleated marrow cells, a sensitive functional assay for stemcell localization was employed.An evaluation of spleen hematopoietic colony formation

(CFU-S), after the intravenous infusion of bone marrow cellsinto lethally irradiated recipients, is often utilized to derive afunctional approximation of the number of stem cells presentwithin a given inoculum (9). The CFU-S assay additionallyreflects the efficiency with which pluripotent stem cells areable to localize in potential sites of engraftment containinghematopoietic inductive microenvironments, following theirintravenous infusion (10). To test the hypothesis that com-petitive inhibition of the asialoglycoprotein receptor wouldenhance engraftment efficiency, we injected varying num-bers of unfractionated nucleated marrow cells (103 or 104)into lethally irradiated recipient mice. Half of the mice con-comitantly received 4 mg of ASF, a dose shown to competi-tively inhibit PNAhi cell uptake by the hepatic asialoglyco-protein receptor (2). Ten days after bone marrow transplan-tation, the recipients were sacrificed, and their spleens weresurgically excised and evaluated for hematopoietic colonyformation. In three separate experiments, the results ofwhich are shown in Table 1, we demonstrated a significant(5- to 10-fold) enhancement of CFU-S formation in the

Table 1. CFU-S formation after competitive inhibition of theasialoglycoprotein receptor

Nucleated marrowGroup cells infused, no. Inhibitor CFU-SA 0 0.6 ± 0.8B 103 1.2 ± 1.5*C 103 ASFt 5.7 ± 4.0D 104 5.6 ± 4.3*E 104 ASFt 16.7 ± 11.0

Lethally irradiated C3H/HeN mice (15 animals per group) wereinfused with the stated number of nucleated marrow cells. Datarepresent the compilation of three independent experiments and areexpressed as colonies per spleen (mean ± SEM) observed 10 daysafter transplantation. Data were pooled since each experiment gavecomparable results.*Group B is statistically different from group C (P < 0.01) and groupD is statistically different from group E (P < 0.01). The P value wascalculated by assuming 35 colonies per spleen in the three spleensfrom group E having confluent colonies.tASF (4 mg) or an equivalent volume of complete medium wasadministered with the infused marrow inoculum.

I I -1

Medical Sciences: Samlowski and Daynes

Dow

nloa

ded

by g

uest

on

Aug

ust 2

1, 2

020

2510 Medical Sciences: Samlowski and Daynes

groups receiving the coinfusion of ASF as compared to ani-mals receiving only the nucleated bone marrow cells. Theseexperiments suggested that a temporary (3-4 hr) asialoglyco-protein receptor blockade causes a significant shift in the tis-sue localization properties of stem cells into compartmentsthat have the potential to support stem cell proliferation.

Additional studies were performed to demonstrate that theobserved increase in CFU-S was specifically due to an inhi-bition of the asialoglycoprotein receptor. Lethally irradiatedmice were pretreated with a 4-mg dose of ASF. A parallelexperimental group received an equivalent amount of the na-tive sialated glycoprotein fetuin. Control animals, which re-ceived no potential inhibitor, as well as mice from the twoexperimental groups were then infused with 2 x 103 or 2 x10' syngeneic marrow cells. The results of this experimentare shown in Table 2. Unlike ASF, fetuin proved to be inef-fective in causing enhanced spleen colony formation. Theresults of this experiment strongly suggested that the in-crease in splenic colonies found in animals given ASF duringtransplantation was specifically related to a temporaryblockade of the asialoglycoprotein receptor. Additional ex-periments demonstrated that the coinjection of asialoeryth-rocytes (produced by incubating syngeneic erythrocyteswith the enzyme neuraminidase) was capable of producing asimilar enhancement of spleen colony formation, unlike nor-mal murine erythrocytes (data not shown).To experimentally test the hypothesis that a temporary

competitive inhibition of the asialoglycoprotein receptor atthe time of the initial localization of the infused bone marrowcells was responsible for the enhanced efficiency of the en-graftment process, two groups of lethally irradiated animalswere injected intravenously with nucleated bone marrowcells. The first group of recipients was given ASF at thesame time as the marrow cells and the second group wasinfused with ASF 4 hr after transplantation. Control groupseither received bone marrow cells without potential inhibitoror were left unreconstituted. Ten days after transplant, allanimals were sacrificed, and the number of CFU-S in eachgroup was quantitated. The results (Table 3) clearly demon-strate that ASF is only effective in enhancing CFU-S forma-tion when given at the same time as the bone marrow cellinocuilum.

Histologic analysis of formalin-fixed spleens and femurs,derived from the various groups of animals in the above ex-periments (Fig. 2), demonstrated a marked difference be-

Table 2. CFU-S formation is specifically enhanced by thecoadministration of asialoglycoprotein

Nucleated marrowGroup cells infused, no. Inhibitor CFU-S

A 2 x 103 4.8 ± 2.6*B 2 x103 ASF 20.0 ± 2.0C 2 x103 Fetuin 9.0 ± 3.5D 2 x 104 11.8 ± 1.1*E 2 x 104 ASF TNTCF 2 x 104 Fetuin 16.4 ± 4.5This CFU-S assay was performed as described, with 6-week-old

C3H mice, with the exception that animals were pretreated 15 minprior to the injection ofmarrow cells with an intravenous infusion ofthe potential inhibitor (4 mg of ASF or 4 mg of fetuin). A seconddose of the inhibitor was administered 21/2 hr after the first dose, ata time when we have shown that the effect of the first dose ofinhibitor would shortly be waning (2). Results are expressed as amean number of spleen colonies ± SEM. TNTC, too numerous tocount (>30-40 confluent colonies per spleen).*Group A is statistically different from group B (P < 0.001) but notfrom group C (P > 0.1). Group D is statistically different fromgroup E (P < 0.001) but not from group F (P> 0.05). The P valuewas calculated by assuming an average of 35 colonies in theconfluent spleens.

Table 3. Bone marrow engraftment efficiency is stronglyinfluenced by the events taking place shortlyafter transplantation

Nucleated marrowGroup cells infused, no. Inhibitor CFU-SA None 0B 2 x 103 2.6 ± 1.9*C 2 x 103 ASF 9.8 ± 3.6*D 2 x 103 ASF 3.3 ± 1.9The CFU-S assay was performed with 6-week-old C3H/HeN

mice. Lethally irradiated animals were given 4m-dosof ASF.Group C received the inhibitor several minutes prior to the infusionof marrow cells; group D received ASF 4 hr after the infusion of themarrow cells.*Group B is statistically different from group C (P < 0.01) and groupC is statistically different from group D (P < 0.05). However,groups B and D are not statistically different from one another (P> 0.10) by the two-tailed t test.

tween tissue taken from animals receiving ASF plus marrowcells versus the tissue taken from control mice given marrowcells alone. Microscopically, animals that received only 2 x103 bone marrow cells with concomitant ASF administrationrevealed greater than twice the spleen colony numbers ex-hibited by controls receiving 2 x 104 bone marrow cellsalone (an apparent 20-fold increase in efficiency). A strikingqualitative difference was also observed in the types of colo-nies present in the spleens taken from control and from liverreceptor-blocked groups. The colony architecture in all testgroups was composed mainly of immature myeloid, ery-throid, and undifferentiated hematologic cells. This findingis similar to that originally described by Till and McCulloch,as well as others (6, 11). However, the spleens taken fromgroups of recipient animals that received ASF plus marrowcells showed additional large colonies that were suggestiveof enhanced megakaryocytic differentiation (Fig. 3). His-tologic analysis of the femurs taken from the animals engraft-ed without the competitive inhibitor showed a hypocellularmarrow cavity, with rare myeloid and erythroid colonies in aparatrabecular or endosteal region. Femurs taken from the

A B

FIG. 2. Hematopoietic colony formation (arrows) in the spleensfrom lethally x-irradiated mice 10 days following the infusion of syn-geneic nucleated bone marrow cells. Histologic sections were pre-pared from formalin-fixed spleens of mice receiving either 2 x 103(A) or 2 x io' (B) bone marrow cells alone. (C) Typical spleen takenfrom an animal that received 2 x i03bone marrow cells along withthe competitive inhibitor ASF. Note the marked increase in both thenumber and size of the hematopoietic colonies present. (Hematoxy-in/eosin; x4.)

Proc. NatL Acad ScL USA 82 (1985)

Dow

nloa

ded

by g

uest

on

Aug

ust 2

1, 2

020

Proc. Natl. Acad. Sci. USA 82 (1985) 2511

-i 8-

C. 6--I4-

v x

O 2 -

-'

4~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~1'I Ii

I'JAb*~~

Lir~AA~ ~~~

FIG. 3. A large megakaryocytic colony observed 10 days afterlethal irradiation and reconstitution with bone marrow cells in thepresence of the competitive inhibitor of the asialoglycoprotein re-ceptor. Similar splenic colonies were not observed in groups receiv-ing nucleated marrow cells alone. (Hematoxylin/eosin; x50.)

groups of mice that were infused with ASF prior to bonemarrow engraftment, however, showed much more exten-sive paraosseous regeneration of marrow, with some infiltra-tion of hematopoietic cells into the intermediate and centralstromal regions. Occasional animals in the group that re-ceived 2 x 104 bone marrow cells plus ASF demonstrated ahypercellular marrow (data not shown). These histologicevaluations of femoral bone marrow seem to correlate withboth CFU-S numbers and morphology, suggesting enhancedmarrow proliferation in anatomic sites that are capable ofsupporting hematopoietic reconstitution.Experiments were next performed to test the survival ad-

vantage following transplantation in the presence of asialog-lycoprotein receptor blockade. Lethally irradiated micewere reconstituted with 2 x 105 nucleated bone marrow cellsin the presence or absence of 4 mg of ASF. Peripheral blood(85 Al) from four animals in each group was assayed for com-plete blood counts by Coulter S+2 at 3- to 4-day intervalsfollowing transplantation. The results of this experiment(Fig. 4) showed a small but consistent increase in the recov-ery rate of both peripheral blood platelet and leukocytecounts in the receptor-blocked group as compared to con-trols. The absolute neutrophil count increased in parallelwith the peripheral blood leukocyte count in both groups.The survival curves for the groups that received ASF plus 2x 105 bone marrow cells showed a significantly lower mor-tality than the control groups that received only marrow cells(84% survival at 35 days, versus 31%, as shown in Fig. 5). By28 days after transplantation, hematopoietic function in bothcontrol and experimental groups had returned to stable lev-els, although these were generally 50-75% of pretransplantbaseline levels.

DISCUSSIONThe procedure of bone marrow transplantation generally in-volves the intravenous infusion of donor bone marrow cellsinto a suitable recipient that is severely immunosuppressed.The pluripotent stem cell population contained within themarrow inoculum must then circulate in the bloodstream, ar-rest, and extravasate into specific tissue sites (such as spleenor marrow cavity). These tissue sites contain stromal ele-ments capable of supporting the engraftment process (1) andhave been termed hematopoietic-inducing microenviron-ments (12, 13). The stem cell population is not generally as-sociated with the circulating pool of blood cells; however,exceptions to this general rule may take place during periods

L-

* .-

So, _

.t XU -u

4 -

3

21

A

B

I I I I I I. I I I I

6 9 12 15 18 21Time, days

FIG. 4. Recovery of leukocyte (A) and platelet (B) counts follow-ing bone marrow transplantation. Lethally irradiated mice were giv-en 2 x 105 bone marrow cells with 4 mg of ASF (*, *) or with nopretreatment (o, A). The groups of animals that were transplanted inthe presence of asialoglycoprotein receptor blockade had 3- to 6-dayearlier peripheral blood leukocyte and platelet count recoveries.

of stress (14, 15). In our experimental system, almost all in-fused marrow cells are associated with tissue sites of local-ization within 3 hr after transplantation, providing supportfor this concept.PNA 'bone marrow stem cells in the circulation are at risk

to encounter the hepatic asialoglycoprotein receptor, whichhas the potential to sequester cells that bear membrane-asso-ciated galactose-terminal glycoconjugates (2). This interac-tion could negatively influence transplantation efficiency,because hematopoiesis does not normally occur within theadult liver (5). It seems possible, therefore, that the numberof infused pluripotent stem cells that will be able to prolifer-ate in the appropriate hematopoietic inductive microenviron-ment might be significantly influenced by a manipulation ofthe initial localization process, which blocks potential hepat-ic localization.The PNAhi phenotype of the bone marrow stem cell popu-

lation may have additional implications to the stem cell local-ization process. Regoeczi et al. have demonstrated the exis-tence of an asialoglycoprotein receptor-like structure in thebone marrow compartment. This bone marrow receptor dif-

100 -

80 -

.> 60-

40-2-20 -

6--

0---

- --

7 14 21Time. days

28 35

FIG. 5. Survival of 16 animal groups that were lethally irradiatedand reconstituted with 2 x 105 bone marrow cells in the presence (e)or absence (a) of ASF. The animals transplanted in the presence ofasialoglycoprotein receptor blockade had a significantly improvedsurvival.

Medical Sciences: Samlowski and Daynes

Dow

nloa

ded

by g

uest

on

Aug

ust 2

1, 2

020

2512 Medical Sciences: Samlowski and Daynes

fers from the hepatic receptor in its enhanced affinity forbiantennary glycans, such as asialotransferrin, rather thantriantennary structures (16). This bone marrow receptormight represent a recognition structure for a galactose-termi-nal ligand on stem cells, which facilitates binding and extrav-asation into the bone marrow cavity. These investigatorsalso demonstrated that certain glycoproteins, such as ASF(which contain predominantly triantennary glycans), have amuch lower affinity for the bone marrow receptor than forthe receptor found in toe liver (16). Therefore, ASF may bean ideal competitive inhibitor because of its potential abilityto block liver localization ofPNAh cells, without significant-ly impairing the potential interaction of the bone marrow re-ceptor with stem cells.Our experiments established that competitive inhibition of

the receptor with ASF or asialoerythrocytes had several ma-jor beneficial effects to bone marrow engraftment efficiency.Splenic and bone marrow colonization by hematopoieticprogenitors was significantly increased when assayed fol-lowing engraftment in the presence of receptor blockade.This increase in the amount of proliferating marrow was ac-companied by a shift in the differentiation of splenic coloniestoward larger megakaryocytic colonies, not usually ob-served in CFU-S assays. The improved efficiency of engraft-ment was further reflected in a somewhat earlier recovery ofleukocyte and platelet counts in mice engrafted with thecoinfusion of ASF. Most important, a significant survivalbenefit was shown in the competitive inhibition group. Fur-ther investigations suggested that the timing of the asialogly-coprotein receptor blockade was critical. When ASF admin-istration was delayed by as little as 4 hr following the iiifu-sion of the bone marrow inoculum, the beneficial effect ofasialoglycoprotein receptor blockade was lost. This observa-tion emphasizes the importance of the stem cell localizationprocess during the initial few hours of transplantation to theeventual outcome of the procedure.The experiments reported herein were performed in a to-

tally syngeneic system to avoid possible interference withengraftment due to graft-versus-host disease. However, sim-ilar experiments have also been performed by utilizing bothallogeneic and semi-allogeneic systems with similar results,suggesting a more widespread applicability (data not shown).Competitive inhibition of the asialoglycoprotein receptormay also be potentially useful in human autologous and allo-geneic bone marrow transplantation, since human stem cellsare known to have a PNA' phenotype (17) and humans alsohave a functioning liver asialoglycoprotein receptor (18). In-clusion of this procedure might therefore be useful in modi-fying the significant morbidity and mortality due to bleedingand infection, which are related to the severe pancytopeniaobserved early in the course of bone marrow transplantation(19).By providing a more efficient engraftment of hematopoiet-

ic progenitor cells, the technique described herein provides apotentially useful adjunct to current attempts to fractionatebone marrow with either cytotoxic antibody (20-22) or lec-tins (23). Both of these procedures have been utilized in anattempt to decrease the incidence of graft-versus-host dis-ease by depleting the donor marrow inoculum of mature Tlymphocytes prior to transplantation. Fractionation tech-

niques, however, are known to significantly reduce the num-ber of stem cells available for infusion (24, 25). The use ofasialoglycoprotein receptor blockade provides a means tooffset this procedural stem cell loss by increasing the per-centage of injected stem cells that is able to localize and pro-liferate in hematopoietic-inducing microenvironments.

This research was supported in part by National Institutes ofHealth Research Fellowship CA 07280, National Institutes of HealthResearch Grants CA 25917 and CA 33065 as well as the ReynoldsFoundation.

1. Tavassoli, M. & Friedenstein, A. (1983) Am. J. Hematol. 15,195-203.

2. Samlowski, W. E., Spangrude, G. J. & Daynes, R. A. (1984)Cell. Immunol. 88, 309-322.

3. Ashwell, G. & Harford, J. (1982) Annu. Rev. Biochem. 51,531-554.

4. Reisner, Y., Itzicovitch, L., Meshorer, A. & Sharon, N. (1978)Proc. Natl. Acad. Sci. USA 75, 2933-2936.

5. Barker, J. E., Keenan, M. A. & Raphals, L. (1%9) J. Cell.Physiol. 74, 51-56.

6. Till, J. E. & McCulloch, E. A: (1961) Radiat. Res. 14, 213-222.

7. Aminoff, D. (1961) Biochem. J. 81, 384-392.8. Spangrude, G. J., Bernard, E. J., Ajioka, R. S. & Daynes,

R. A. (1983) J. Immunol. 130, 2974-2981.9. London, J., Berrih, S. & Bach, J.-F. (1978) J. Immunol. 121,

438443.10. Till, J. E., McCulloch, E. A. & Siminovitch, L. (1964) Proc.

Natl. Acad. Sci. USA 51, 29-36.11. Curry, J. L. & Trentin, J. J. (1%7) Dev. Biol. 15, 395-413.12. Trentin, J. J. (1971) Am. J. Pathol. 65, 621-628.13. Lambertsen, R. H. & Weiss, L. (1984) Blood 63, 287-297.14. Tavassoli, M. & Yoffey, J. M. (1983) Bone Marrow: Structure

and Function (Liss, New York), pp. 239-251.15. Neuwirt, J. (1982) in Recent Advances in Hematology, Immu-

nology and Blood Transfusion, ed. Hollan, S. R. (Wiley, NewYork), pp. 117-132.

16. Regoeczi, E., Chindemi, P. A., Hatton, M. W. C. & Berry,L. R. (1980) Arch. Biochem. Biophys. 205, 76-84.

17. Galili, U., Galili, N., Or, R. & Polliack, A. (1981) Clin. Exp.Immunol. 43, 311-318.

18. Baenziger, J. U. & Maynard, Y. (1980) J. Biol. Chem. 255,4607-4613.

19. Winston, D. J., Gale, R. P., Meyer, D. V., Young, L. S. & theUCLA Bone Marrow Transplantation Group (1979) Medicine58, 1-31.

20. Thierfelder, S., Hoffman-Fezer, G., Rodt, H., Doxiadis, I.,Eulitz, M. & Kummer, U. (1983) Transplantation 35, 249-254.

21. Spruce,W. E., McMillan, R., Miller, W., Fox, R., Carson, D.,Schwartz, D. B., Hartman, G. A., Renshaw, L. W. & Beutler,E. (1983) Transplantation 36, 369-372.

22. Reinherz, E. L., Geha, R., Rappeport, J. M., Wilson, M.,Penta, A. C., Hussey, R. E., Fitzgerald, K.. A., Daley, J. F.,Levine, H., Rosen, F. S. & Schlossman, S. F. (1982) Proc.Natl. Acad. Sci. USA 79, 6047-6051.

23. O'Reilly, R. J., Kapoor, N., Kirkpatrick, D., Cunningham-Rundles, S., Pollack, M. S., Dupont, B., Hodes, M. Z., Good,R. A. & Reisner, Y. (1983) Transplant. Proc. 15, 1431-1435.

24. Reisner, Y., Kapoor, N., Kirkpatrick, D., Pollack, M. S., Du-pont, B., Good, R. A. & O'Reilly, R. J. (1981) Lancet i, 327-331.

25. Reisner, Y., Kapoor, N., O'Reilly, R. J. & Good, R. A. (1980)Lancet ri, 1320-1324.

Proc. NatL Acad Sci. USA 82 (1985)

Dow

nloa

ded

by g

uest

on

Aug

ust 2

1, 2

020