doi:10.1182/blood-2002-03-0896Prepublished online August 29, 2002;2003 101: 576-584   

 Schipul, Jr and Paul W. KincadeMaria Isabel D. Rossi, Takafumi Yokota, Kay L. Medina, Karla P. Garrett, Philip C. Comp, Arthur H. developmental age-related changesB lymphopoiesis is active throughout human life, but there are

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IMMUNOBIOLOGY

B lymphopoiesis is active throughout human life, but there are developmentalage-related changesMaria Isabel D. Rossi, Takafumi Yokota, Kay L. Medina, Karla P. Garrett, Philip C. Comp, Arthur H. Schipul Jr, and Paul W. Kincade

This study addressed several questionsconcerning age-related changes in hu-man B lymphopoiesis. The relative abun-dance of pro-B, pre-B, immature, naive,and mature B cells among the CD19�

lymphocyte fraction of human bone mar-row was found not to change appreciablyover the interval between 24 and 88 yearsof age. Moreover, proliferation of pro-Band large pre-B cells in adult marrowequaled that observed with fetal marrowspecimens. Exceptionally low numbersof lymphocyte precursors were found insome marrow samples, and the valuesobtained were used to determine parame-

ters that best reflect B lymphopoiesis.Cord blood always contained higher inci-dences of functional precursors than adultcells. However, sorted CD34� Lin� CD10�

progenitors from cord blood and adultmarrow had equivalent potential for differ-entiation in culture, and notable age-related changes were found in more primi-tive subsets. A recently described subsetof CD34�CD38�CD7� cord blood cellshad no exact counterpart in adult marrow.That is, all adult CD34�Lin�CD7�CD10�

cells expressed CD38, displayed lessCD45RA, and had little B-lineage differen-tiation potential. The CD7� fractions in

either site contained progenitors for ery-throid and natural killer (NK) lineages,and ones sorted from marrow expressedhigh levels of transcripts for the CD122interleukin 2 (IL-2)/IL-15 receptor requiredby NK-lineage precursors. Dramaticchanges in human B lymphopoiesis oc-cur early in life, and more information isrequired to construct a probable se-quence of differentiation events prior tothe acquisition of CD10. (Blood. 2003;101:576-584)

© 2003 by The American Society of Hematology

Introduction

Humoral immune responses are known to be compromised in theaged, and decreased numbers of B and T lymphocytes parallelincreases in natural killer (NK) cells.1-3 Animal studies haverevealed age-related changes in B-lymphocyte production withinbone marrow (BM).4-6 Numbers of pro-B cells that can be resolvedby flow cytometry are relatively stable with age, but pre-B cellsdecline substantially.4,7 Incorporation of bromodeoxyuridine (BrdU)into dividing cells indicates that B-cell production is diminished inold mice.7 This corresponds to the reduced entry of newly formedcells to the long-lived lymphocyte pool.7,8 Functional precursoractivity may decline because of a diminished support capacity bymarrow stromal cells.4,9 All of these observations indicate thatperipheral B-lymphocyte populations in the aged may be replen-ished with fewer cells from marrow, with potential consequencesfor the repertoire of available antibody specificities. However, it isunclear if such findings are completely applicable to humans.

Animal studies have also been informative about molecularmechanisms and steps in B lymphopoiesis. Rare hematopoieticstem cells transit a number of “compartments” that are defined onthe basis of cell surface markers. In reality, this represents acontinuum where genes are expressed and repressed in a temporal,but not necessarily synchronous, fashion. Early precursors havemultiple differentiation options and may express transcriptionfactors in a way that does not reflect their ultimate fate.10,11 Cellswith potential for differentiation to lymphoid, but not myeloid or

erythroid, cells have been referred to as common lymphoidprogenitors.12,13 These Lin�c-kitloTdT�IL-7R��/lo precursors likelyderive from early lymphoid progenitors in the very rare Lin�c-kithiSca-1hiCD27� fraction of BM cells.14,15 However, it is impor-tant to note that the differentiation pathways used during fetal lifemay be quite different from adult BM.16-18

Knowledge of early B-lymphocyte progenitors in humans lagsbehind that for mice, and there are important differences. Interleu-kin 7 (IL-7) is essential to this process in mice but not in humans,and no combination of cytokines has been identified that supportsvigorous human lymphocyte formation in culture.19-21 Indeed, themost effective models for human B lymphopoiesis use murinestromal cells that provide uncharacterized stimuli.22 Cytokines andhormones have been identified that selectively inhibit B-lympho-cyte formation in mice.13,23-27 It is important to know if lymphopoi-esis is altered as humans age because such negative factorsincrease. Some investigative techniques are not appropriate withspecimens of human marrow, and the yield of hematopoietic cells isvariable. Cellularity decreases with age in some but not all humanbones,28,29 and most studies have been performed on marrowaspirates contaminated by peripheral blood.30,31

Despite these species differences and technical difficulties,progress is being made in understanding the process in humans. Forexample, evidence indicates that CD10�CD19� cells may be thehuman equivalent of common lymphoid progenitors in the mouse,

From the Immunobiology and Cancer Program, Oklahoma Medical ResearchFoundation; Department of Medicine, University of Oklahoma Health SciencesCenter; and St Anthony Hospital, Oklahoma City.

Submitted March 21, 2002; accepted August 14, 2002. Prepublished online asBlood First Edition Paper, August 29, 2002; DOI 10.1182/blood-2002-03-0896.

Supported by grants AI 20069 and AI 45864 from the National Institutes ofHealth. P.W.K. holds the William H. and Rita Bell Chair in biomedical research.

M.I.D.R. and T.Y. contributed equally to this work.

Reprints: Paul W. Kincade, Immunobiology and Cancer Program, OklahomaMedical Research Foundation, 825 NE 13th St, Oklahoma City, OK 73104;e-mail: kincade@omrf.ouhsc.edu.

The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.

© 2003 by The American Society of Hematology

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whereas candidates recently identified in umbilical cord blood(CB) are CD34�CD38�CD7�.32,33 A major goal of this study wasto determine if numbers and functions of such precursors declinewith age because that could reflect important changes in humanlymphopoietic capacity.

Expression of Rag genes in marrow of mature individualsmight suggest that immunoglobulin genes are being rear-ranged.34,35 Unfortunately, little information has been availableabout proliferation36,37 or differentiation activity in humans, andit is possible this slows with age. Thus, although the incidenceof lymphocytes has been known to decline between birth andpuberty, there was an insufficient understanding of changes thatmay occur subsequently.31,35,38

Ratios of precursor subsets in human marrow, proliferativeindices, and differentiation were evaluated in the present study withthe goal of identifying age-related changes. We now report thatwhereas B-cell precursors are depressed in some patients, the entiredifferentiation series is normally present throughout life and ismitotically active. However, surface marker changes occur early inlife, and adult marrow has reduced potential for lymphocyteproduction in 2 model systems. Additional information aboutacquisition of CD7 and CD10 may prove helpful in understandingthe sequence of early differentiation events in humans.

Materials and methods

Cell sources

The CB samples were obtained from placentas of healthy newbornscollected at the Oklahoma University or St Anthony hospitals (OklahomaCity). Adult marrow samples were collected from patients between 24 and88 years of age who were undergoing hip replacement surgery at the Boneand Joint Hospital (Oklahoma City, OK). Patient records with no identify-ing information were used to determine that some individuals had a recenthistory of treatment with anti-inflammatory drugs such as prednisone ornaproxen. Most specimens came from otherwise healthy patients with noevidence of malignancy or immunosuppression. The Anatomic Gift Founda-tion (Laurel, MD) supplied femoral BM from fetuses at 16 to 24 weeks ofgestation. Investigational Review Boards at Oklahoma Medical ResearchFoundation and The University of Oklahoma Health Sciences Centerapproved all procedures involving human specimens.

Animals

NOD/LtSz-scid/scid (nonobese diabetic/severe combined immunodefi-ciency [NOD/SCID]) mice were obtained from a breeding colony estab-lished at the Oklahoma Medical Research Foundation (OMRF; OklahomaCity, OK) from breeding pairs kindly provided by Dr Leonard D. Schultz(Jackson Laboratory, Bar Harbor, ME). Animals were housed in a restrictedbarrier facility under specific pathogen-free conditions.

Antibodies and cytokines

The following antibodies were either purified, biotinylated, or conjugatedwith fluorescein isothiocyanate (FITC), phycoerythrin (PE), or allophyco-cyanin (APC): anti-CD38 (clone HIT2), anti-CD24 (clone ML5), anti-CD23 (clone M-L233), anti-CD45 (clone HI30), anti–Ki-67 (clone B56),anti-IgD (clone IA6-2) from BD-Pharmingen; (San Jose, CA), anti-CD34(HPCA-2), anti-CD3 (clone UCHT1), anti-CD7 (clone 6B7), anti-CD8(clone HIT8a), anti-CD14 (clone TUK4), anti-CD16 (clone 3G8), anti-CD10 (clone 5-1B4), anti-CD19 (clone SJ25-C1), anti-CD13 (cloneTUK1), anti-CD27 (clone CLB-27/1), anti-CD33 (clone 4D3), anti-CD56(clone NKI nbl-1), anti-CD64 (clone 10.1), anti-� light chain (cloneHP6062), anti-� light chain (clone HP6054), and anti–glycophorin A (cloneCLB-ery1) from Caltag Laboratories (Burlingame, CA). Fab� fragments ofgoat antihuman IgM or antihuman IgD were from Southern Biotechnology

Associates (Birmingham, AL). The biotinylated antibodies were revealedwith streptavidin red 613 (Gibco BRL, Rockville, MD). Purified recombi-nant human (rh) erythropoietin (rhEPO), rh stem cell factor (rhSCF), rhFlt3ligand, rhIL-15, and rh granulocyte colony-stimulating factor (rhG-CSF)were purchased from R & D Systems (Minneapolis, MN).

Cell isolation and staining procedure for fluorescenceactivated cell sorting analysis

Four-color immunofluorescence analysis was used for the identification ofthe different B-cell precursor populations in total nucleated BM cellsuspensions from fetal and adult marrow. Briefly, single-cell suspensionswere obtained by flushing or gently vortexing the BM. After staining,erythrocytes were lysed using the Becton Dickinson lysis buffer solutionaccording to the manufacturer’s instructions and CD45 antibody was usedto assess the percentages of leukocytes in each sample. Intracellularstaining was done by permeabilizing cells after surface staining. The cellswere fixed with 1% paraformaldehyde in phosphate-buffered saline (PBS)and permeabilized with 70% ethanol at �20°C, for more than 30 minutesand washed twice with the staining buffer. They were then incubated withantihuman IgM for 30 minutes at room temperature. To establish theproliferative fraction, cells were incubated with the antibody anti-Ki67(mib-1) for 1 hour on ice, as described by Zupo et al.39 Data acquisition andanalyses were performed on a FACSCalibur (Becton Dickinson).

Isolation of CD34�/� cells and cell sorting

Mononuclear cells were collected from CB or adult BM by Ficoll/Hypaque(Lymphocyte Separation Medium; Cellgro-Mediatech, Herndon, VA) cen-trifugation. Enrichment for CD34� cells was performed using the DirectCD34 Isolation Kit (Miltenyi Biotec, Auburn, CA). CD34�-enriched cellswere stained with CD34-FITC, lineage markers (Lin, CD13, CD14, CD33,and CD64 for myeloid cells; glycophorin A for erythroid cells; CD19 for Bcells; CD3 and CD8 for T cells; CD56 for NK cells)–PE, CD7-biotinfollowed by streptavidin red 613, and CD10-APC. CD34�Lin�CD10�CD7�,CD34�Lin�CD10�CD7�, and CD34�Lin�CD10�CD7� cells were sortedusing a MoFlo (Cytomation, Fort Collins, CO). The sorted cells weresubsequently subjected to flow cytometry, culture, and gene expressionanalyses. In some experiments, CD34�CD38� cells were sorted andcultured. In this case, mononuclear cells were incubated with mouseantihuman antibodies: CD3, CD13, CD14, CD16, CD33, CD56, andglycophorin A, and negatively selected using the BioMag goat antimouseIgG-coated beads (Perseptive Biosystems, Framingham, MA). The en-riched cells were stained with CD34-FITC and CD38-PE. CD34�CD38�

cells were determined using gating previously described.40 Analysis aftersorting revealed the purity of the sort populations to be more than 95%.

Transplantation of human cells into NOD/SCID mice

Magnetically enriched CD34� cells from CB or fetal or adult BM werediluted in PBS with 0.1% bovine serum albumin (BSA fraction V; SigmaChemical, St Louis, MO) and injected into the tail vein of sublethallyirradiated (100 cGy from a 137Cs source) 8- to 12-week-old male or femaleNOD/SCID mice. Then, 5 � 106 CD34� CB mononuclear cells werecoinjected as a source of accessory cells.41,42 No chimerism was everobtained in mice given transplants of the accessory cells alone.

LDAs

The frequencies of B-cell progenitors in CB and adult BM were determinedby plating CD34�CD38� cells in limiting dilution assays (LDAs). Between20 and 180 wells containing pre-established MS-5 stromal cell layers (akind gift of Dr J. Mori, Niigata University) were plated with 3, 5, 10, 15, 20,30, 50, 75, 100, 150, or 200 cells each using the automated cell depositionunit of the MoFlo. Cells were cultured in �-minimal essential medium(MEM; Cellgro-Mediatech) supplemented with 10% fetal calf serum (FCS;Hyclone, Logan, UT) and a combination of rhSCF (100 ng/mL) andrhG-CSF (10 ng/mL).21 Wells were inspected every week for the presenceof new clones. Positive wells were harvested after 5 to 7 weeks of cultureand analyzed by flow cytometry for CD19� cells. The frequencies of

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precursors were calculated by linear regression analysis on the basis ofPoisson distribution as the reciprocal of the concentration of test cells thatgave 37% negative cultures.

Multilineage coculture system and methycellulose culture

Sorted CD34�Lin�CD10�CD7�, CD34�Lin�CD10�CD7�, and CD34�

Lin�CD10�CD7� cells were cultured at 1 � 104 cells/6 mL/25-cm2 tissueculture flask (Corning, Corning, NY) with MS-5 stromal cells. Culturemedia was �-MEM containing 10% FCS, rhSCF (100 ng/mL), rhG-CSF(10 ng/mL), rhFlt-3 ligand (100 ng/mL), and rhIL-15 (10 ng/mL). Thecultures were maintained with weekly whole medium changes and thegeneration of myeloid, B, and NK cells was evaluated at the indicatedperiods. Methylcellulose cultures were performed in 35-mm plates (Corn-ing) using MethoCult GF H4534 medium (Stem Cell Technologies,Vancouver, BC, Canada) containing rhSCF, rh granulocyte-macrophagecolony-stimulating factor (rhGM-CSF), and rhIL-3 along with 3 U/mLrhEPO (R & D Systems). All cultures were maintained at 37°C in ahumidified incubator with 5% CO2 in air. Differential colony counts werescored after 10 to 14 days by morphologic characteristics using an invertedmicroscope and confirmed by staining individual colonies with Wright orMay-Grunwald-Giemsa stain.

RT-PCR

Sorted cells were put in lysis buffer (Ambion, Austin, TX) and mRNA wasextracted using Poly-A column (Ambion) according to the manufacturer’sinstructions. cDNA was prepared from mRNA treated with DNase I usingoligo-dT and Moloney murine leukemia virus reverse transcriptase (RT;Gibco BRL). Semiquantitative polymerase chain reaction (PCR) was doneto measure relative differences in transcript levels of target cDNAs againstlevels of the reference gene GAPDH. The PCR was done in 100 �Lcontaining PCR buffer (Takara Biomedical, Osaka, Japan), 1.5 mM MgCl2,200 �M deoxyadenosine triphosphate (dATP), deoxyguanosine triphos-phate (dGTP), deoxythymidine triphosphate (dTTP), 50 �M deoxycytidinetriphosphate (dCTP), and 50 pmol of each primer. For quantification, 0.5�Ci (0.0185 MBq) [�32P] dCTP (Amersham, Arlington Heights, IL) wasincluded in each reaction tube. Samples were denatured in a DNAthermocycler (Perkin-Elmer, Norwalk, CT) for 10 minutes at 95°C. Toincrease specificity, 2.5 U Taq DNA polymerase (Takara) was added to eachsample during this initial denaturation. Samples were then cycled for 1minute at 94°C, 1 minute annealing 60°C, 1.5 minutes extension at 72°Cwith a final extension of 7 minutes at 72°C. Aliquots were removed atcycles 25, 28, 31, and 34 for glyceraldehyde phosphate dehydrogenase(GAPDH) and cycles 32, 35, and 38 for all others to ensure that PCRremained within the exponential range of amplification. Then, 5-�Laliquots were denatured in a formamide loading buffer and applied to a 6%polyacrylamide gel containing 7 M urea. Incorporation of [alpha32P] dCTPinto PCR product bands was quantified from dry gels using a PhosphoIm-ager (Molecular Dynamics, Sunnyvale, CA).

Primers were as follows: GAPDH sense: 5�-TCCAAAATCAAGT-GGGGCGAT-3�, GAPDH antisense: 5�-TTCTAGACGGCAGGTCAGGTC-3�, 475-bp expected product; RAG1 sense: 5�-CCTGAGTCCTCTCATT-GCTGAGAG-3�, RAG1 antisense: 5�-AGGGCATGATGATCGCCATACT-3�,681-bp product; RAG2 sense: 5�-CTAATGAAGAGCAGACAACATTCA-3�, RAG2 antisense: 5�-TAGGACTCTTTGGGGAGTGTGTAG-3�, 422-bpproduct; EBF sense: 5�-CCGGGCTCACTTTGAGAAGCAG-3�, EBF anti-sense: 5�-CAGGGAGTAGCATGTTCCAGAT-3�, 638-bp product; Pax5sense: 5�-CTCGGTGAGCACGGATTCGGCC-3�, Pax5 antisense: 5�-GCGGCAGCGCTATAATAGTAG-3�, 621-bp product; CD122 sense: 5�-GCGTGGCTCGGCCACCTC-3�, CD122 antisense: 5�-GACGATGAGGG-GAAGGGCGAAGA-3�, 211-bp product, Id-1 as described.43

Statistical analysis

All results are shown as mean values SD. Differences between groupswere assessed using the Student t test.

Results

B lymphopoiesis persists throughout adult human life

It has been reported that absolute numbers of B-lymphocyte precursorsin human marrow decline with normal age and particularly duringadolescence.38 Therefore, we calculated percentages of CD19�sIg�

(surface Ig) lymphocytes and CD19�sIg� B cells relative to totalnumbers of nucleated cells in our adult marrow samples (data notshown). As in the previous study,38 sample-to-sample variations wereconsiderable, but we saw no substantial or consistent depletion of theselymphocyte populations as a function of age in adults. We then extendedprevious studies31,35 describing B-lineage lymphocyte precursors inadult human marrow (Figure 1A). Pro-B cells (CD34�CD10�CD19�),pre-B cells (CD34�CD19�sIg� or CD19�c��sIg�) and immature Bcells (sIgM�CD24hiIgD�CD10�CD38�) represented relatively con-stant percentages of the CD19� lymphocytes over a 24- to 88-yearage range. The ratios between these populations and the presence ofnaive (sIgMhiIgD�CD24hiCD10�/�CD38�/�CD27�) B cells wouldall be consistent with stable B lymphopoiesis. Mature recirculatingB cells (sIgM�IgD�CD24loCD38�) were previously shown tocomprise a large fraction of marrow lymphocytes.35 We found nomature B cells in fetal bones, but there was no substantial influenceof adult age on this population (Figure 1A). Mature B cells inmarrow were also CD27�, a characteristic of somatically mutatedmemory B cells in peripheral blood.44 Most female marrow donorswere receiving estrogen replacement therapy, and we found nosex-related differences in this analysis.

Blood cell production results from massive division withinmarrow, and we assessed expression of the Ki-67 nuclear prolifera-tion antigen in B-lineage lymphocyte precursors (Figure 1B).Again, we found no age-related changes in this parameter andmale-versus-female values were comparable. A similar analysiswas recently performed with fetal marrow specimens,45 and the

Figure 1. Relative frequencies and mitotic activity of B-lineage precursorsin BM do not change during adult life. (A) The incidences of pro Bcells (CD34�CD10�CD19�), pre-B cells (CD19�sIgM�c��), immature B(IgM�CD24hiCD10�IgD�), naive B (IgM�CD24hiIgD�CD38�/�), and mature B(IgM�CD24loIgD�CD38�) cells were determined by flow cytometry and shown aspercentages of total viable CD19� lymphocytes (%) SDs. (B) Expression of theKi-67 nuclear proliferation antigen was determined by flow cytometry, using low-anglelight scatter to resolve large and small lymphocytes. The data represent averagepercentages of pro-B cells (CD34�CD19�) and pre-B cells (CD19�sIgM�c��) SDs. Differences between groups were not statistically significant (P .05).

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results are included here for comparison (Figure 1B). With theexception of small pre-B cells, which are more mitotically activeduring embryonic life, B-cell precursors were remarkably similarwith respect to Ki-67 expression. We conclude that activelyproliferating precursors are present within the lymphocyte fractionof human marrow until at least the eighth decade.

Analysis of marrow from exceptional patients reveals usefulindices of B-cell production

The present data would be consistent with stable B lymphopoiesisoccurring over a large age range. However, it is difficult to knowthe degree to which lymphopoietic marrow is replaced by fat as afunction of age and to control for sample-to-sample variations withrespect to peripheral blood contamination and location. Therefore,it would be helpful to have other parameters for assessing marrowactivity. Relatively constant proportions of B-lineage lymphocyteswere found in most of the specimens we examined (Figure 1A).Whenever immature (IgM�CD24hiIgD�CD10�CD38�) B cellswere present, marrow samples contained mitotically active pre-B(CD19�sIgM�) cells identified with the Ki-67 antibody. However,exceptionally low numbers were found in samples obtained fromsome patients. There were insufficient numbers of these individualsand information about specific treatments to formally assign themto groups. However, the degree of sample-to-sample variation inthese specimens provided an opportunity to seek correlations(Figure 2). In these individuals, incidences of cycling pre-B cellspredicted the presence of immature lymphocytes (P � .0001),consistent with a close precursor-product relationship betweenthem. Naive (IgM�CD24hiIgD�CD38�CD10�/�D27�) B cells didnot closely correlate with proliferating pre-B cells (P � .1035),although they are the progeny of immature B cells. We emphasizethe usefulness of Ki-67 expression because simple percentages ofpre-B cells did not correspond as closely with numbers of newlyformed lymphocytes (data not shown). The findings suggest thatthe size of the proliferating pre-B cell pool determines numbers ofnewly formed B cells.

Early developmental age-related changes in populationsresolved by flow cytometry

Earlier reports demonstrated that CD7 could be used to discrimi-nate hematopoietic stem cells from more differentiated precursorsof NK and T lymphocytes.46,47 It is also noteworthy that CD7 israrely coexpressed with CD10 or CD19, and evidence indicates

that CD7� cells in CB represent common lymphoid progenitors.33

However, comparisons made by flow cytometry suggested that CBmight not be representative of differentiation pathways used inadult marrow. Adult CD34�Lin� cells contained a more conspicu-ous subset of CD7�CD10� progenitors as well as moreCD7�CD10� cells (Figure 3A). Additional differences were foundby comparing sorted CD7� and CD7� subsets of CD34�Lin�CD10�

cells from the 2 sources (Figure 3B-C). It has been suggested thatCD45RA is acquired before CD10,32 and we found that nearly halfof the CB CD7� cells expressed moderate to high levels (Figure3B). In contrast, only 10% of the comparable subset from adultmarrow was CD45RA�. As noted above, the CD38� subset of theCD34�Lin�CD7� fraction of CB has been recently found tocontain multilymphoid but not myeloerythroid progenitors.33 It is

Figure 3. Early lymphohematopoietic progenitors in adult BM differ from onesin CB with respect to surface marker characteristics. (A) CD7/CD10 profiles areshown for CD34�Lin� cells in CB and adult BM. CD34� Lin�CD10�CD7� andCD34�Lin�CD10�CD7� cells were sorted from CB and adult BM and stained withCD45RA-PE (B) or CD38-PE (C). These data are representative of 3 independentlysorted samples.

Figure 2. Profiles of immature and naive B cells in somedeficient marrow specimens reveal a correlation be-tween cycling pre-B cells and immature B cells. Fluo-rescence-activated cell sorter (FACS) profiles of immature(IgM�CD24hiCD38�IgD�) and naive (IgM�CD24hiCD38�/�IgD�)B cells from a healthy patient (A-B) are compared to a deficientone (C-D). Immature B cells were also CD10� (not shown). Theindex of cycling pre-B cells in exceptional patient samplescorrelated with production of immature B cells (E), and lessclosely with naive B cells (F). Coefficients of determination (R2)are indicated.

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remarkable that cells with that combination of markers do not existin adult marrow (Figure 3C). Note that the CD7� subsets of fetaland adult populations were similar with respect to CD45RA andCD38 expression. Thus, the composition of hematopoietic cellsubsets or surface marker display in CB differs substantially fromadult BM.

Transplantation experiments reveal age-related changesin differentiation potential

Immunodeficient NOD/SCID mice were prepared for transplanta-tion with low-dose irradiation (“Materials and methods”) and theninjected with highly enriched suspensions of CD34� cells (Table1). Previous studies documented an extraordinary differentiationpotential of CB stem cells in this model,48-50 so we compared onelog less CD34� cells from this source to ones isolated from fetaland adult marrow. The latter represented pools of CD34� cellsobtained from specimens ranging from 35 to 82 years of age in oneexperiment and 62 to 77 years of age in a second transplantation.Flow cytometry was performed on aliquots of the suspensions todetermine numbers of CD34�CD38� cells injected because ofreports that these cells are particularly effective for engraftment ofNOD/SCID mice51 (Table 1). It has been shown that undefinedaccessory cells can influence engraftment of human cells inmice.41,42 Therefore, we added 5 � 106 CD34� CB mononuclearcells to all samples of CD34� cells before transplantation, and therecipients were assessed 7 weeks later. No human cells weredetectable in animals that received only the CD34� cells, but easilydiscernible populations of CD45�CD19� lymphocytes were foundin virtually all recipients of CD34� cells, regardless of donor age(Table 1). Adult precursors were less effective than those obtainedfrom the other 2 sources with respect to numbers of lymphocytesproduced, suggesting a possible consequence of the early develop-mental age-related changes noted.

Culture experiments reveal age-related differencesin expansion and differentiation potentialof lymphocyte progenitors

Homing to the marrow, resistance to xenotransplantation barriers,and many other factors could influence the degree of chimerismobserved in the NOD/SCID model. Furthermore, culture experi-ments could permit detailed analysis of small cell numbers.Therefore, we performed an LDA in which graded numbers ofCD34�CD38� cells were seeded onto monolayers of murine MS-5stromal cells and stimulated with SCF and G-CSF (Table 2). Thefrequency of precursors with the potential for clonal expansionvaried from 3.6% to 9.78% in marrow suspensions obtained fromhealthy 24- to 88-year-old donors. In separate experiments, thecloning efficiency of umbilical CB CD34�CD38� cells rangedfrom 11.11% to 14.29% using the same culture conditions. In thiscase, the B-cell progenitor frequency was 6.67% (range, 5.56%-8.33%). Furthermore, in about 40% of the wells more than 50% ofthe cells produced by CB CD34�CD38� cells were CD19�, and

this was never the case with adult BM. Analysis of pooled clonesrevealed that differentiation did not proceed efficiently beyond theCD19� stage in such cultures and very few cytoplasmic or surface�� cells were recovered.

Culture conditions were then developed that allowed simulta-neous differentiation of CD19� B- lineage, CD13/CD33� myeloid-lineage, and CD56� NK-lineage cells (“Materials and methods”).Absolute numbers of CD19� cells produced were equal to or morethan those obtained with other methods we have tried andNK-lineage cells were always efficiently produced when IL-15 waspresent. The method was used for a side-by-side comparison offetal/neonatal-versus-adult marrow precursors. Three-week cul-tures initiated with 104 CD34�Lin�CD7�CD10� CB precursorsyielded much larger numbers of B-lineage lymphocytes than thoseprepared with equivalent cells from adult BM (Figure 4A).Interestingly, the lymphoid potential of CD34�Lin�CD7�CD10�

cells from the sources was comparable. The CD34�Lin�CD7�CD10�

subset was efficient only when derived from CB. All subsets of CByielded approximately 10 times the number of CD56� NK-lineage cellsthan obtained in cultures of their adult counterparts.

Myeloid potential was highest in the CD34�Lin�CD7�CD10�

fractions and dramatically down-regulated in CD7� or CD10�

subsets, regardless of source. These results are consistent withclonal assays conducted with methylcellulose cultures. With 2analyses of CB, most nonlymphoid progenitors were present in theCD7� subset (710.8 74.6 colonies/1000 cells). However, it isnoteworthy that an average of 170.8 50.8 colonies/1000 cellswere also obtained when the CD7� fraction was plated and 95% ofthem were erythroid. Similarly, the CD34� Lin�CD7�CD10�

fraction of adult marrow contained most (an average of 60% in 3experiments) of the granulocyte/macrophage progenitors (granulo-cyte/macrophage colony-forming units [CFU-GMs]). As with CBcells, 98% of the colonies obtained with the CD34�Lin�CD7�

fraction were erythroid (erythroid burst-forming units [BFU-Es]).Insufficient numbers of the rare CD7�CD10� subset of adultmarrow precluded their analysis in these cultures.

Weekly examination revealed that myeloid progeny appearedquickly and then declined in cultures initiated with the CD34�

Lin�CD7�CD10� fraction of CB, whereas myelopoiesis was morepersistent in cultures of their adult counterparts (Figure 4B-C).

Table 2. Adult human BM contains functional B-lineage precursors that can beenumerated in limiting dilution cultures of CD34�CD38� cells

Source ofCD34�CD38� cells

Clonogenic frequency,% (CI)

Frequency of cells able togive rise to CD19� cells, % (CI)

CB 12.5 (11.11-14.29) 6.67 (5.56-8.33)

Adult BM, 24 y 6.09 (4.84-13.53) 1.16 (0.77-1.96)

Adult BM, 44 y 9.78 (7.78-19.01) 1.04 (0.67-1.79)

Adult BM, 45 y 3.69 (2.78-6.56) 0.05 (�0.01-0.12)

Adult BM, 70 y 4.5 (3.82-8.83) 0.18 (0.07-0.31)

Adult BM, 88 y 3.6 (3.24-4.26) 0.15 (0.02-0.32)

CI indicates confidence interval.

Table 1. Adult human BM contains B-lymphocyte precursors revealed by transplantation in immunodeficient NOD/SCID mice

Cells injected No. of cells InjectedNo. of CD34�CD38�

cells injected*No. of engrafted/injected

mice (%)Percentage of hCD45�CD19�

cells in BM of chimeras

Fetal BM CD34� 2.9-3 � 106 28.5-30 � 103 5/6 (83) 1.54 ( 1.28)

CB CD34� 5 � 105 4 � 103 11/11 (100) 1.02 ( 0.86)

Adult BM CD34� 2 � 106 39.6-72 � 103 5/5 (100) 0.22 ( 0.16)

CB CD34� 5 � 106 �0.01 0/5 �0.02

*Numbers of CD34�CD38� cells were determined by flow cytometry, using small aliquots of each preparation of CD34� enriched cells. These results were obtained in 2very similar but independent experiments.

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Progressive production of B- and NK-lineage cells was consistentwith the primitive nature of progenitors in this fraction. Whereastransient B-lineage differentiation was found with theCD34�Lin�CD7�CD10� fraction, this was never the case with thecomparable adult marrow subset. The adult population also yieldeda wave of NK-lineage cells as contrasted with sustained productionfrom neonatal cells.

Neonatal or adult CD10� fractions contained B-lineage precur-sors that differentiated within 2 weeks in culture, and CD56�

NK-lineage cells reached peak values 1 week later. This analysisrevealed large age-related changes in the potential of highlypurified CB and adult BM progenitors to expand in culture.

CD10 and CD7 expression denote commitmentto B and NK lineages

These findings highlight the importance of learning more about theB-lineage precursors in human marrow. Therefore, adult subsets sortedfor use in the culture experiments as described were also subjected toRT-PCR analysis (Figure 5). Transcripts associated with B-lymphocytelineage differentiation (RAG1, RAG2, EBF, and Pax5) were markedlyup-regulated in the CD34�Lin�CD10�CD7� population relative to themore primitive CD34�Lin�CD10�CD7� subset. Furthermore, levels ofthe Id-1 transcriptional repressor were reduced in that fraction (data notshown). In contrast, expression of the CD122 receptor for IL-15/IL-2required by NK-lineage cells was strongly associated with display ofCD7. Although transcripts for RAG1 and RAG2 were detectable inCD7� cells, this fraction was deficient in the EBF transcriptionfactor required for B lymphopoiesis. Very similar results wereobtained in 3 independent experiments. These results complementthose obtained with culture experiments and suggest that acquisi-tion of CD10 or CD7 corresponds to substantial progression in theB or NK lineages, respectively.

Discussion

This study was informative about several aspects of human Blymphopoiesis. Ratios between precursors and immature B cellswithin marrow and an indication of mitotic activity were found tobe remarkably constant throughout adult life. Exceptional marrowsamples from some patients were deficient in lymphocyte precur-sors, providing a unique opportunity to see how the abundance ofparticular subsets corresponds to newly formed B cells. Althoughthe potential for lymphoid differentiation, as assessed by cultureand transplantation assays was retained in most individuals, thelymphocyte yield from adult progenitors was much less than thatobtained from CB. Furthermore, we documented age-relatedchanges in subsets resolved on the basis of CD7 and CD10expression. Analysis of highly purified progenitors revealed thaterythroid and NK differentiation potential might diverge fromB-lineage potential as cells acquire one or the other of these markers.

There are many technical problems associated with analysis ofhuman marrow specimens, and it is difficult to estimate absolutenumbers of B-cell precursors. As one example, mature recirculat-ing B cells have been identified that have a distinctive density of

Figure 5. B-lineage characteristics are established by the CD10� stage in adult BM.Expression of lineage-related genes was examined in the CD34�Lin�CD10�CD7� (f),CD34�Lin�CD10� CD7� (o), and CD34�Lin�CD10�CD7� (�) fractions sorted from adultBM. RT-PCR was used to amplify transcripts for the indicated genes, and the results werenormalized according to GAPDH expression.

Figure 4. Early lymphohematopoietic progenitors in CB and adult BM differwith respect to expansion and differentiation potential. CD34�Lin�CD10�CD7�,CD34�Lin�CD10�CD7�, and CD34�Lin�CD10�CD7� cells were sorted from CB oradult BM and cultured with MS-5 stromal cells (1 � 104 cells/flask in each fraction).Yields of CD19� B–, CD56� NK–, and CD13�/CD33� myeloid–lineage cells in3-week cultures are shown (A). The B-, NK-, and myeloid-lineage compositions werealso determined weekly in cultures initiated with fractions sorted from CB (B) or adultBM (C). Similar results were obtained in 2 independent side-by-side comparisonsbetween CB and adult BM.

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CD24 and sIgM.35 Absolute numbers of these cells per unit marrowor body weight are not known, but they progressively dilute B-cellprecursors and newly formed B cells. However, it is fortunate thatother measurements reflect the lymphopoietic activity in that organ.As in previous studies31,35 the ratios between B-cell precursors andnewly formed B cells can be determined, and we confirm that theyare relatively constant. Moreover, the proliferative activity ofB-cell precursors was surprisingly stable with age. In a separatestudy, we evaluated precursors in fetal marrow and found that anexceptionally large fraction of small pre-B cells in that site areKi-67�.45 This is one of several distinctive features of fetallymphopoiesis. Once the process is established in adult marrow, theproliferation index of B-cell precursors remains unchanged until atleast 88 years of age.

The striking deficiency of B-cell precursors in some of themarrow specimens suggests that influences of therapy and environ-mental stress on B lymphopoiesis merit further study. One previousreport recorded delayed recovery of B cells after marrow transplan-tation in patients receiving cortisone.52 Most of our deficientsamples came from patients who were being treated with anti-inflammatory drugs and we will show elsewhere that humanlymphocyte progenitors are very sensitive to glucocorticoids. Herewe used the variation provided by these unusual specimens to makeinferences about precursor-product relationships within marrow.The best correlation was found between cycling pre-B cells andimmature B cells. The Ki-67 proliferation antigen provided aparticularly useful index, and simple pre-B cell percentages did notcorrelate as well. Whenever percentages of proliferating pre-B cellswere beneath 50%, immature (IgM�IgD�CD24hiCD10�CD38�) Bcells were scarce. The correlation between percentages of naiveCD38� B cells and cycling pre-B cells was not as good as therelationship between pre-B cells and immature B cells (Figure 2).Cells with a naive phenotype might randomly leave marrow toenter the peripheral B-cell pool, as has been suggested for newlyformed B cells in mice.53

It was important to learn if cells with surface characteristics ofB-cell precursors actually had the potential to give rise to lympho-cytes. Human CD34�CD38� cells have been determined in otherstudies to be responsible for engraftment of NOD/SCID mice, butuncharacterized accessory cells among the CD34� fraction ofhematopoietic cell suspensions can influence the outcome.41,42,51 Tominimize this parameter, we added a constant number of CBCD34� mononuclear cells to each of the transplant samples, andhuman CD19� cells were easily detectable in marrow of all micereceiving transplants. As others have shown, stem cells withinCB are exceptionally efficient for engraftment of immunodefi-cient mice.48-50 We estimate that 7 weeks after transplantationthere were 111 98 human CD19� cells present in each mousefemur per injected CD34�CD38� CB cell. This value is 10- to100-fold higher than we obtained with precursors isolated fromadult marrow.

Limiting dilution cultures initiated with highly purifiedCD34�CD38� cells provided an independent means of assessingdifferentiation potential and one that should be less influenced bycontaminating accessory cells. CB precursors had a higher cloningefficiency in vitro than adult marrow–derived cells, and more of theresponding wells generated CD19� lymphocytes (Table 2). Thisresult is compatible with observations made in another study, usinga different culture system54 and suggests the possibility of anintrinsic difference between neonatal and adult progenitors.

Because the proportion of hematopoietic marrow relative to fatprogressively declines with age in long bones, total numbers of

newly formed B cells might be reduced in some locations.28

However, the cellularity of marrow in other sites is remarkablyconstant throughout life, and it will be important to determine thetotal body output of new B cells.29 This important issue awaitsother types of analyses comparable to those recently used to assessthymic activity in adult humans.55-57 Many studies suggest thathematopoietic stem cells retain the potential for replenishinglymphocytes throughout life.58,59 However, there are changes withrespect to incidence, homing potential, and function on a per-cellbasis. Additionally, there can be age-related declines in the abilityof environmental cells within marrow to support lymphopoiesis.4

Genetic polymorphisms influence stem cell numbers and senes-cence to a substantial degree.59,60 For that reason, our culture assayswere conducted with cells from individual marrow donors. Al-though flow cytometry revealed proportions of lymphocyte precur-sors in most marrow specimens to be remarkably constant,functional precursor frequencies among CD34�CD38� cells werehighly variable (Table 2).

Flow cytometry experiments revealed that the composition andpatterns of surface marker expression differ substantially betweenCB and adult BM. The interesting CD34�CD38�CD7�CD10�

subset recently described in cord blood33 has no direct counterpartin adults. Furthermore, the CB CD34�CD38�CD7�CD10� subsetdiffered substantially from the comparable adult population withrespect to expression of CD45RA. Given these changes, we electedto sort 3 populations for side-by-side comparisons in culturestudies. CD10�CD19� cells comprise a very small proportion(2.5% 1.1%) of the 1% of marrow mononuclear cells thatexpress CD34�30,32 (Figure 3). Most of these CD34�CD10�CD19�

cells also express TdT30,32,61 and data not shown) and already haveD-JH rearrangements.34 Previous reports32,34 suggest that thisfraction is a rich source of early B-lineage precursors. Our cultureanalyses substantiate that conclusion and it is noteworthy that CBand adult progenitors were equivalent in potential for B-lineagedifferentiation (Figure 4). PCR analysis of CD34�Lin�CD7�CD10�

cells from adult BM revealed substantial expression of 4 genesrequired for B lymphopoiesis (Figure 5). However, it is alsoimportant to stress that CD10� cells have some myeloidpotential that can be appreciated in long-term cultures but not inclonal assays32 (Figure 4; data not shown). Thus, a completelylymphoid-restricted progenitor has not been isolated from adulthuman marrow.

The question arises whether B lineage–associated events beginprior to the CD10� stage and CD7� cells are interesting in thisregard. One recent study found that a CD34�CD38�CD7� fractionof CB contains what might be the human equivalent of commonlymphoid progenitors described in mice.12,33 We confirm that CBCD34�CD7�CD10�CD19� cells can rapidly generate small num-bers of CD19� cells in culture (Figure 4; data not shown). Unlikethe previous report, we detected some nonlymphoid, and especiallyerythroid, precursors in this population. This may be because wedid not additionally select for CD38� cells in CB. However, asnoted, there are no CD38�CD7� cells in adult human marrow andthe total CD34�Lin�CD7�CD10� fraction of marrow was a poorsource of B-lineage progenitors (Figures 3-4). PCR analysis ofcarefully sorted cells substantiated the culture results andshowed a considerable enrichment for cells expressing theCD122 receptor for IL-15/IL-2 required for NK-lineage differen-tiation (Figure 5). It was previously reported that theCD34�CD7� fraction of adult marrow contains precursors ofNK cells.46 Thus, CD7 expression may characterize an interme-diate stage between stem cells, erythroid cells, and pro-B cells in

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neonates, but be more closely associated with NK lineage–restricted precursors in adult marrow.

To summarize, several distinctive adult characteristics of B-lymphocyte precursors may be acquired during the neonatal or adoles-cent period. The primitive CD34�Lin�CD10�CD7� fraction retainsdifferentiation potential throughout life, but it is much less efficient thanCB cells with the same phenotype. Cells bearing CD10 include many

early B-lineage precursors and ones in neonates and adults appear tohave equivalent differentiation potential. All subsequent stages ofB-lineage differentiation are represented in constant ratios in marrowand numbers of proliferating pre-B cells closely correlate with numbersof newly formed B cells. The search must continue for lymphoid-restricted progenitors in human marrow and our understanding of earlydifferentiation events is incomplete.

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