Bone Marrow Transplantation, (1998) 21, 79–84 1998 Stockton Press All rights reserved 0268–3369/98 $12.00

Cadaveric bone marrow and spleen cells for transplantation

G Soderdahl1, C Tammik2, M Remberger2 and O Ringde´n1,2

Departments of1Transplantation Surgery and2Clinical Immunology, Huddinge Hospital, Huddinge, Sweden

Summary:

Cadaveric bone marrow was harvested from 20 brain-dead donors to determine optimal conditions for pro-curement for transplantation. The number of nucleatedcells obtained from 1 ml of bone marrow wassignificantly higher in vertebrae (876 203 106,mean6 s.e.m.) than in the sternum (10.26 3.83 106)or ribs (4.9 6 2.03 106). Viability of cells was not sig-nificantly affected by storage temperature (48C or 208C)or duration of storage (6–72 h). In addition to bone mar-row, spleen cells were harvested from three cadavericdonors. The mean yield from 1 g of spleen tissue was4.43 106 nucleated cells. Using magnetic beads, weremoved 96% of T lymphocytes without affecting thetotal yield of stem cells from cadaveric bone marrow.Using CD34-positive cell selection, we obtained a 99.6%T cell depletion efficiency, but with a loss of 60% ofCD34-positive cells. Using optimized techniques, weobtained an estimated mean yield of 5.53 1010 mono-nuclear cells from the whole thoracic and lumbar ver-tebral column. With a mean fraction of CD34-positivecells of 2.16 0.3%, recovery and purity were not affec-ted by site of sample, temperature or donor age. In con-trast, the CD34-positive fraction in spleen preparationswas 0.416 0.06%. When analyzing the number ofcolony-forming units (CFU-GM, BFU-E and CFU-GEMM), we found no significant differences betweencadaveric bone marrow and bone marrow aspiratesfrom living donors. However, cells harvested from thespleen gave significantly fewer CFUs than did bonemarrow from living donors. We conclude that bonemarrow from cadaveric donors can be harvested andprocured with a high degree of viability and good func-tion. With an appropriate technique of harvesting andprocurement, it seems feasible to recover enough stemcells for transplantation.Keywords: bone marrow; spleen cells; transplantation;cadaveric; hematopoietic stem cells

In the field of bone marrow transplantation, only one thirdof candidates for allogeneic bone marrow transplantationhave an HLA-identical sibling donor.1 Therefore, marrowfrom unrelated donors is increasingly employed.2–4 Bonemarrow from cadaveric donors used as a source may be an

Correspondence: Dr O Ringde´n, Dept of Clinical Immunology, HuddingeHospital, F79, S-141 86 Huddinge, SwedenReceived 27 June 1997; accepted 31 August 1997

alternative to volunteer donors. In addition, bone marrow,harvested together with solid organs, can even be used tomodulate the immune response of the organ recipient. Thediscovery of microchimerism after transplantation of solidorgans has been considered to explain mechanisms of long-term allograft acceptance without immunosuppression.5–7

Thus, donor-specific bone marrow augmentation after solidorgan transplantation has been thought to promote extendedchimerism and possibly to produce tolerance.8,9 Toleranceinduction has been described with living donor leukocyteinfusions in mice,10 dogs11 and rhesus monkeys.12 Hitherto,extension of these results to human transplantations hasfailed to induce tolerance although improved renal graftsurvival was seen.13

Several methods for harvesting cadaveric bone marrowhave been described.14,15 However, little is known aboutoptimization of the number of stem cells required for suc-cessful engraftment in the recipient. This paper describes arapid method for the harvesting and procurement ofcadaveric bone marrow. Isolation conditions have also beenstudied to optimize the yield of progenitor cells. Finally,the quality of the processed cadaveric bone marrow hasbeen analyzed and compared with bone marrow aspiratedfrom living donors.

Materials and methods

Bone marrow was taken from 20 cadaveric organ donors.In five, kidneys only were taken, in eight cases, liver and/orpancreas were also taken and in the remaining seven, tho-racic organs were removed as well. Informed consent wasgiven by relatives. Around 70% of those approached gaveconsent. University of Wisconsin (UW) solution16 was usedfor organ preservation in all cases. The median age of thedonors was 47 years with a range between 4–67 years (ninemales and 11 females). The cause of death in seven caseswas trauma and the other 13 deaths were due to intracranialhemorrhage. For comparison, bone marrow aspirated fromthe iliac crest of 10 living donors, median age 28 years (7–61 years), eight males and two females was analyzed.

Bone marrow harvest

Bone marrow was harvested from vertebral bodies, sternumbody and ribs. In three cases, sternal samples were takenbefore perfusion of organs with UW solution. To obtainmarrow from vertebral bodies, the outer cortex wasremoved with an electrical bone saw and the trabecularbone containing red marrow was extracted, using a Giglisaw or a chisel. Trabecular samples were cut into small

Cadaveric bone marrowG Soderdahl et al

80pieces and transported to the laboratory in heparinizedRPMI 1640 medium either on ice (+4°C) or at roomtemperature (+20°C). For sternal and rib samples, wholesections of bone marrow were removed with an electric sawand thereafter cut into smaller pieces.

Bone marrow procurement

Samples were analyzed immediately or stored for amaximum of 3 days at room temperature (+20°C) or in arefrigerator (+4°–+8°C) before separation. Cell suspensionswere passed through a sterile 70-mm cell strainer (Falcon;Becton Dickinson, San Jose, CA, USA) to remove bonefragments before layering on a Ficoll–Hypaque gradientand then centrifuged at 500g for 20 min. The interfacecontaining mononuclear cells was collected and the cellswere washed and resuspended in RPMI 1640, sup-plemented with 10% human AB serum. Viability of bonemarrow cells was tested with trypan blue exclusion.

Spleen cell procurement

Spleens were taken from cadaveric organ donors, then cut,emulsified through a cell strainer and washed.17 The cellswere separated on a Ficoll–Hypaque gradient and were keptfrozen in liquid nitrogen before use.

FACS analysis of cell surface markers

The method for FACS analysis on Ficoll-separated cellshas been described previously.18 Briefly, after cells werecounted, they were diluted in PBS to 106 cells/ml. To 0.1ml cell suspension, 10ml of conjugated monoclonal anti-bodies (mabs) were added; CD45 FITC/CD14 PE(Leucogate; Becton Dickinson), r1/r2a Simultest Control(Becton Dickinson), CD34 FITC (HPCA-2; BectonDickinson) and CD19 PE (HD-37; Dako, Copenhagen,Denmark), CD3 FITC (UCHT-1, Dako A/S) and CD 56 PE(Leu-19, Becton Dickinson). Cell suspensions were incu-bated for 10 min, at room temperature in the dark. Afterwashing once and resuspension in PBS, the cells were ana-lyzed in a FACSort (Becton Dickinson), evaluating 10–20 000 cells per sample. After running the negative control,100 ml propidium iodide (1:1000) were added to the cellsand after incubation for a further 5 min, the cells were ana-lyzed to evaluate viability. The percentages of the variouscell populations were calculated in an ungated cell popu-lation.

Assay for hematopoietic precursors

A commercial kit ‘Stem cell CFU kit’ (GIBCO BRL, LifeTechnology, NY, USA) was employed to study colony for-mation and was used according to the manufacturer’sinstructions. This semi-solid system mimics the extracellu-lar matrix produced by stromal cells. Triplicate cultureswith 12.53 103 cells/well, 0.5 ml in each well, were incu-bated for 10 days. Colonies were identified as BFU-E(burst-forming units–erythroid cells) containing denselypacked hemoglobinized cells, CFU-GM (colony-formingunits–granulocytes, macrophages) and CFU-GEMM

(colony-forming units–granulocytes, erythroid cells, macro-phages, megakaryocytes).

T cell depletion

Prepared bone marrow cells were resuspended in RPMI+2% FCS to a final concentration of 1–4× 107/ml. Magneticbead (Dynal, Oslo, Norway)-coupled mabs directed againstCD2, CD3 and CD8 were added to a mean of four beads/Tcell and incubated for 30 min at+4°C on a rocking plat-form. Cells with surface-bound beads were removed witha magnetic device and the supernatant containinguncoupled cells was collected. This procedure was perfor-med twice to achieve maximal clearance of magnetic beads.

CD34-positive cell selection

For CD34 cell selection a commercially available systemwas used according to the manufacturer’s instruction(Ceprate, LC-34; Cellpro, Bothell, WA, USA). In this sys-tem, cells are incubated with a biotin-labelled anti-CD34antibody and labelled target cells are captured with avidin-coated beads, using a continuous flow column. Bound tar-get cells are released by gentle agitation.

Results

Immune markers and hematopoiesis

Table 1 shows the fractions of various populations of hema-topoietic cells, assayed by FACS analysis. The number ofcolony-forming units in the analyzed samples is alsoshown. Three of the sternal samples were collected in anearly phase of the donor operation before procurement oforgans and perfusion with UW solution. However, therewere no differences in viability, yield of nucleated cells orcell populations in these samples as compared to thosetaken from the sternal body at the end of the donor oper-ation. The fraction of CD34-positive cells was not affectedby temperature or storage for less than 3 days (Figure 1).Patients above 47 years of age had 2.06 0.4 (6 s.e.)%CD34-positive cells in the marrow, as compared to2.26 0.4 in donors below this age (NS).

Viability and yield

The viability of mononucleated cells immediately aftergradient centrifugation ranged between 84–99%, with amean value of 93.56 1.0% (mean6 s.e.m.). No significantdifference was observed between samples from the ver-tebrae, rib or sternum (data not shown). Viability was notsignificantly affected by storage temperature or duration ofstorage (maximum 3 days), although we found a tendencyto decreased viability when samples were stored for a pro-longed period at room temperature (Figure 2).

The yield of nucleated cells per ml of bone marrow wasfound to be higher from vertebrae than from sternal bodyor rib (P , 0.001) (Figure 3). The mean yield from onevertebral body was calculated as 2.66 0.73 109 mono-nuclear cells. Since samples from vertebral bodies proved

Cadaveric bone marrowG Soderdahl et al

81Table 1 Fraction of immune markers and number of progenitors of bone marrow from various sources

Fraction of mononucleated cells % No. of colonies/105 cells

n CD3 CD34 CD19 CD56 n CFU-GM BFU-E CFU-GEMM

Vertebrae 17 7.0± 0.8 2.1± 0.2 5.4± 0.5 0.6± 0.1 11 373± 91 560± 114 67± 36Sternuma 7 7.1± 1.5 2.1± 0.4 3.9± 1.0 1.3± 0.6 3 642± 274 394± 216 144± 8Sternumb 3 5.0± 0.6 1.9± 0.7 ND 2.2± 1.9 — ND ND NDRib 3 8.3± 3.1 3.0± 0.3 4.8± 0.9 0.9± 0.4 1 200 136 96Spleen 3 21.1± 1.7 0.4± 0.1 ND ND 3 99± 49 192± 91 3.0± 2.9LD 10 11.2± 0.5 1.3± 0.3 4.2± 0.5 1.4± 0.1 10 638± 102 708± 260 128± 32

ND = not done; LD= living donors.The results are expressed as mean± s.e.m.aSamples taken after perfusion of organs with UW solution.bSamples taken before perfusion of organs with UW solution.

2.8

2.6

2.4

2.2

2.0

1.8

1.6

1.4

1.2Day 1 Day 3

Refr, n = 3RT, n = 3

CD34-positive cell kinetics

% C

D34

Day 2

Figure 1 CD34-positive cells (mean6 s.e.) in cadaveric bone marrowstored for 1 to 3 days in a refrigerator (+4 to +8°C) K, and at room tem-perature (+20°C) k.

100

95

90

85

80Day 1 Day 2 Day 3

RefrRT

%

Figure 2 Effect of temperature and duration of storage on viability ofnucleated cells. The results are expressed as mean6 s.e.m. RT= roomtemperature (+20°C), n = 5; refrigerator (REFR)= +4 to +8°C, n = 4.

to be superior as regards yield, a calculation of the maximalyield from one donor was performed. This was based onthe harvest of 10 vertebral bodies, each containing 30 g ofbone marrow with a mean yield of 8.73 107 mononucle-ated cells/ml. This resulted in a total number of 2.63 1010

mononuclear cells and 5.53 108 CD34-positive cells.

100

80

60

40

20

0

×106

P < 0.001

Vertebrae Sternum Rib Spleen

Figure 3 Yield of mononuclear cells/g from various sources. Results areexpressed as mean× 106. Vertebrae,n = 17; sternum,n = 7; rib, n = 3;spleen,n = 3.

Colony-forming units

During the analysis for colony-forming units (CFU-GM,BFU-E and CFU-GEMM), no significant differences wereseen when samples were stored for 3 days on ice (+4°C)or at room temperature (+20°C) (Figure 4). When the num-ber of colony-forming units from cadaveric bone marrowwas compared with preparations of bone marrow from liv-ing donors, there were no significant differences betweeneither of the assays (Table 1).

T cell depletion

Depletion with anti CD3, CD4 and CD8-coupled magneticbeads, to remove T lymphocytes, reduced the total numberof mononucleated cells by 146 3.5%, with no detectableloss of CD34-positive cells. The fraction of CD3-positivecells decreased from 8.66 4.1% to 0.366 0.23%, whichindicates a 966 2.0% depletion efficiency. When T cellswere depleted with the Ceprate method of selection forCD34-positive cells, 99.6% of the T cells were depleted,but with a loss of 60% of the CD34-positive cells as well(Table 2).

Cadaveric bone marrowG Soderdahl et al

82

1200

1100

1000

900

800

700

600

500

400

300

200

100

0

BFU-E

CFU-GEMM

CFU-GM

Day 1RT

Day 1+4°C

Day 2RT

Day 2+4°C

Day 3RT

Day 3+4°C

Co

lon

ies/

105

cel

ls

Figure 4 Effect of temperature and duration of storage on the number of colony-forming units. The results are expressed as mean of three tests. RT= room temperature; or refrigerator= +4 to +8°C.

Table 2 Comparison of T cell depletion using magnetic beads (Dynabeads) with the use of CD34-positive cell selection (Cellpro CC-34)

Dynabeads Cellpro CC-34

Before After Yield % Before After Yield %

No. of nuclear cells 156± 3.3 137± 7.0 84 250 2.8 1.3No. of CD34+ cells 2.5 2.5 100 3.6 1.3 40CD3+ cells 13.0 0.4 3 13.0 0.1 0.9

The number of cells are expressed as mean× 106 ± s.e.m.

Spleen cell preparations

The fractions of the various hematopoietic cells analyzedby flow cytometry are shown in Table 1. The fraction ofCD34-positive cells was significantly lower in spleen cells,as compared to bone marrow harvested from vertebral bod-ies (P , 0.003). There were also fewer CFUs in spleencell preparations than in bone marrow from living donors(CFU-GM; P = 0.02). The mean yield from 1 g of spleentissue was 443 106 mononuclear cells. Assuming a meanweight of 100 g, the total obtainable number of CD34-positive cells from a single spleen would be estimated at193 106.

Discussion

It was earlier shown that cadaveric vertebral bodies are asuitable source of bone marrow for allogeneic transplan-tation.14 The method described includes resection of thethoracolumbar column en bloc for further processing in thelaboratory. In this paper, we describe a modified methodwhich leaves intervertebral discs and adjacent cortical bonein place to give additional strength for the column, which

is advantageous for ethical reasons. This method is rela-tively fast and easily performed after removal of theabdominal organs allowing excellent exposure of the thora-covertebral column.

The mean number of nucleated cells obtained from a ver-tebral body is 8.7× 107/ml bone marrow, which is some-what lower than has earlier been reported. However, someof the samples taken at the beginning of the series showedextremely low values, probably because of technical diffi-culties with procurement and inexperience in the harvestingprocedure. Using data from the 10 most recent donors, atotal yield per donor of 6.23 108 CD34-positive cells wasobtained which seems sufficient for transplantation. A pointof practical value is that the loss of cells during the first36 h is not a crucial problem and the start of procurementcan be postponed until the day after the donor operation.Between 1 and 43 106 CD34 cells are sufficient forengraftment using unrelated peripheral blood progenitorcells.19 Only a few CD34 cells are lost during cryopreser-vation.20 Thus, 6.23 108 CD34 cells would be enough fortwo adult recipients with 70 kg body weight, giving4 3 106 CD34-positive cells/kg.

An interesting question arises when comparing cadaveric

Cadaveric bone marrowG Soderdahl et al

83donor (CD) bone marrow with bone marrow aspirated fromliving donors (LD). We saw no differences in viability ofthe cells after the procurement. The fact that the fractionof CD34-positive cells was smaller in the LD marrow thanin the CD marrow probably indicates that aspirated marrowis diluted with peripheral blood. When quality, as measuredby colony-forming units, of CD marrow is compared withbone marrow aspirated from living donors, we see a tend-ency towards lower amounts in the cadaveric marrow, butthe difference is not significant.

For conventional allogeneic bone marrow transplan-tation, the establishment of registers of voluntary livingdonors has become an important alternative to HLA ident-ical siblings in the search for a well-matched graft.2–4 How-ever, another potential source is cadaveric bone marrow,procured from organ donors, then frozen and stored forfuture transplantations.

In Sweden, the annual number of organ donors is about15 per 106 population. If bone marrow were to be routinelyharvested and stored, a bank acquiring 120 cases a yearcould be established. As compared to the four milliondonors available in various registers, it can be argued thatthis number is negligible, but due to local variations in themost common HLA patterns, it could provide an additionalsource when searching for a well-matched graft, forinstance in ethnic minorities. As regards costs, the methodfor harvest and procurement of cadaveric bone marrow isrelatively simple and fast, when used together with organretrieval. There is a not inconsiderable cost for freezing andstorage. However, it should be noted that no additionalcosts would be incurred by HLA-typing and viral screening,since these are done in any case for organ donors. We there-fore think that the procurement of cadaveric bone marrowfrom brain-dead donors is justified.

It does not seem worth storing the spleen to be used forstem cell transplantation because an average spleen con-tains only 1.93 107 CD34+ cells. This would give a doseof only 0.33 106 CD34+ cells/kg to an average adult.

In organ transplantation, despite recent advances inimmunosuppression, rejection remains the most commoncause of allograft failure, and immunosuppressive agentsare a significant cause of morbidity and mortality in clinicaltransplantation. The possibility of tolerance induction andavoidance of long-term immunosuppression would rep-resent a major step in the development of organ transplan-tation. Donor-specific bone marrow infusion, as an adjunctto solid organ transplantation, has been observed to pro-mote microchimerism and induction of tolerance in animalmodels. In man, such infusions have also been associatedwith establishment of chimerism andin vitro evidence ofdecreased antidonor reactivity in mixed lymphocyte cul-tures, and so far only one study showed an improvementin graft survival.5,6,13 However, in none of the trials haspreconditioning of the patient prior to bone marrowinfusion been performed. The reason for this is fear of tox-icity, neutropenia, infections and severe GVHD due toHLA-incompatibility.

T cell depletion of transplanted bone marrow has provedto be effective in preventing acute GVHD in HLA-identicalsiblings.21,22 If T cell depletion is combined with conven-tional immunosuppressive agents, the GVHD reaction, even

in non-HLA identical matches, may be controlled when acombination of allogeneic and T cell-depleted autologousmarrow is given after ablative therapy.7,23,24 This wouldthen seem to be a possible way to achieve ‘macrochimer-ism’ and tolerance towards the HLA antigens of the infusedbone marrow. Time constraints do not permit giving abla-tive and immunosuppressive conditioning prior to organtransplantation. Furthermore, there may be very high risksof infections if organ and bone marrow transplantation areperformed simultaneously. We would therefore suggest thatconditioning and bone marrow transplantation is delayedfor some weeks until the surgical wounds have healed andthe patient is in a stable clinical condition.

We conclude that bone marrow from cadaveric donorsfor transplantation can be procured with a high degree ofengraftment potential. Our data indicate that the yield ofstem cells is better when marrow is taken from vertebraeinstead of from the ribs or sternal body. With this tech-nique, it seems possible to isolate a sufficient number ofthe cells required for bone marrow transplantation.

Acknowledgements

This study was supported by grants from the Children’s CancerFoundation (1994-060), the Swedish Cancer Foundation (0070-B95-09XCC), the Belve´n Foundation, the Swedish MedicalResearch Council (B96-16X-05971-16C), the FRF Foundationand the Tobias Foundation.

References

1 Thomas ED, Storb R, Clift RAet al. Bone-marrow transplan-tation. Parts I and II.New Engl J Med1975; 292: 832–843and 895–902.

2 Marks DI, Cullis JO, Ward KNet al. Allogenic bone marrowtransplantation for chronic myeloid leukemia using sibling andvolunteer unrelated donors. A comparison of complications inthe first 2 years.Ann Intern Med1993;119: 207–214.

3 Ringden O, Remberger M, Persson Uet al. Similar incidenceof graft-versus-host disease using HLA-A, -B and -DR ident-ical unrelated bone marrow donors as with HLA-identical sib-lings. Bone Marrow Transplant1995;15: 619–625.

4 Downie TR, Hows JM, Gore SMet al. A survey of use ofunrelated donor bone marrow transplantation at 46 centersworldwide, 1983–93. International Unrelated Search andTransplant (IMUST) Study.Bone Marrow Transplant1995;15: 499–503.

5 Rao AS, Thomson AW, Shapiro R, Starzl TE. Chimerism afterwhole organ transplantation: its relationship to graft rejectionand tolerance induction.Curr Opin Nephrol Hypertens1994;3: 589–595.

6 Wren SM, Nalesnik M, Hronakes MLet al. Mixed chimerismto induce tolerance for solid organ transplantation.J PediatrSurg 1991;26: 439–443.

7 Sykes M, Sachs DH. Mixed allogenic chimerism as anapproach to transplantation tolerance.Immunol Today1988;9: 23–27.

8 Fontes P, Rao AS, Demetris AJet al. Bone marrow aug-mentation of donor-cell chimerism in kidney, liver, heart, andpancreas islet cell transplantation.Lancet1994;344: 151–155.

9 Shapiro R, Rao AS, Fontes Pet al. Combined kidney/bone

Cadaveric bone marrowG Soderdahl et al

84 marrow transplantation – evidence of augmentation of chimer-ism. Transplantation1995;59: 306–309.

10 Monaco AP, Wood ML. Studies on heterologous antilympho-cyte serum in mice. VII. Optimal cellular antigen for inductionof immunologic tolerance with antilymphocyte serum.Trans-plant Proc1970;2: 489–496.

11 Caridis DT, Liegeois A, Barrett I, Monaco AP. Enhanced sur-vival of canine renal allografts of ALS-treated dogs givenbone marrow.Transplant Proc1973;5: 671–674.

12 Thomas FT, Carver FM, Foil MBet al. Long-term incompat-ible kidney survival in outbred higher primates withoutchronic immunosuppression.Ann Surg1983;198: 370–378.

13 Barber WH, Mankin JA, Laskow DAet al. Long-term resultsof a controlled prospective study with transfusion of donor-specific bone marrow in 57 cadaveric renal allograft recipi-ents.Transplantation1991;51: 70–75.

14 Rybka WB, Fontes PA, Rao ASet al. Hematopoietic progeni-tor cell content of vertebral body marrow used for combinedsolid organ and bone marrow transplantation.Transplantation1995;59: 871–874.

15 Mugishima H, Terasaki P, Sueyoshi A. Bone marrow fromcadaver donors for transplantation.Blood 1985;65: 392–396.

16 Southard JH, Belzer FO. Organ preservation.Annu Rev Med1995;46: 235–247.

17 Ringden O. Activation of human lymphocyte subpopulationsby rabbit anti-human b2 microglobulin and bylipopolysaccharide.Scand J Immunol1976;5: 891–900.

18 Loken M. Immunofluorescence techniques. In: Molamed MR,Lindmo T, Mendelsohn ML (eds).Flow Cytometry and Sort-ing, 2nd edn. New York: Wiley-Liss, 1990, p 341.

19 Ringden O, Potter MN, Oakhill Aet al. Transplantation ofperipheral blood progenitor cells from unrelated donors.BoneMarrow Transplant1996;17 (Suppl. 2): S62–S64.

20 Ek S, Ringde´n O, Markling L et al. Effects of cryopreser-vation on subsets of fetal liver cells.Bone Marrow Transplant1993;11: 395–398.

21 Prentice HG, Blacklock HAA, Janossy Get al. Depletion ofT lymphocytes in donor marrow prevents significant graft-versus-host disease in matched allogeneic leukaemic marrowtransplant recipients.Lancet1984; i: 472–475.

22 Ringden O, Pihlstedt P, Markling Let al. Prevention of graft-versus-host disease with T cell depletion or cyclosporin andmethotrexate. A randomized trial in adult leukemic marrowrecipients.Bone Marrow Transplant1991;7: 221–226.

23 Sykes M, Chester CH, Sachs DH. Protection from graft-versus-host disease in fully allogenic chimeras by prior admin-istration of T cell-depleted syngenic bone marrow.Transplan-tation 1988;46: 327–330.

24 Moses RD, Orr KS, Bacher JDet al. Cardiac allograft survivalacross major histocompatibility complex barriers in the rhesusmonkey following T-lymphocyte-depleted autologous marrowtransplantation. II Prolonged allograft survival with extensivemarrow T cell depletion.Transplantation1989;47: 435–444.