Proc. Natl. Acad. Sci. USAVol. 92, pp. 10302-10306, October 1995Immunology

The purification and characterization of fetal liver hematopoieticstem cellsSEAN J. MORRISON, HOuMAN D. HEMMATI*, ANTONI M. WANDYCZ*, AND IRVING L. WEISSMANtDepartments of Pathology and Developmental Biology, Stanford University, Stanford, CA 94305

Contributed by Irving L. Weissman, June 28, 1995

ABSTRACT Thy-1°oSca-l+Lin-Mac-l+CD4- cells havebeen isolated from the livers of C57BL-Thy-1.1 fetuses. Thispopulation appears to be an essentially pure population ofhematopoietic stem cells (HSC), in that injection of only sixcells into lethally irradiated adult recipients yields a limitdilution frequency of donor cell-reconstituted mice. Sixty-seven to 77% of clones in this population exhibit long-termmultilineage progenitor activity. This population appears toinclude all long-term multilineage reconstituting progenitorsin the fetal liver. A high proportion of cells are in cycle, andthe absolute number of cells in this population doubles dailyin the fetal liver until 14.5 days postcoitum. At 15.5 days post-coitum, the frequency of this population falls dramatically.Long-term reconstituting HSC clones from the fetal liver giverise to higher levels of reconstitution in lethally irradiatedmice than long-term reconstituting HSC from the bone mar-row. The precise phenotypic and functional characteristics ofHSC vary according to tissue and time during ontogeny.

The developmental and self-renewal potentials of mouse he-matopoietic progenitors can be tested at a clonal level in vivo(1). Long-term self-renewing and transiently self-renewingmultipotent progenitors can be independently purified fromadult mouse bone marrow based on differences in cell surfacemarker expression (2-4). These populations include all mul-tipotent progenitors in the strain of mouse studied (5) andappear to form a lineage from the earliest isolatable stem cellto the most differentiated multipotent progenitor prior tolineage commitment (unpublished data).The mammalian fetal liver contains hematopoietic stem

cells (HSC) capable of long-term multilineage reconstitutionof adults (6, 7). The existence of fetal liver progenitors capableof long-term self-renewal and clonal multilineage reconstitu-tion was unambiguously demonstrated in retroviral markingexperiments in mice (8-10). Thy-1lOLin-/lOSca-1± cells, rep-resenting -0.05% of mouse fetal liver, are highly enriched formultipotent progenitors (refs. 11 and 12; K. Ikuta, personalcommunication). Long-term reconstituting multipotent pro-genitors have also been greatly enriched from mouse fetal liverby isolating AA4.1+Linlo (8) or AA4.1+LinloSca-1+ cells (13).AA4.1+LinloSca-1+ cells represent only 0.05-0.08% of fetalliver cells, but the relative representations of long-term recon-stituting clones and other progenitors within this populationwere not reported (13).We set out to purify mouse fetal liver HSC to determine

whether multipotent progenitors segregated into populationsphenotypically and functionally similar to those described inmouse bone marrow.

MATERIALS AND METHODSMouse Strains. The C57BL/Ka-Thyl.1 (Ly5.2 and Ly5.1

strains) and C57BL/J-Ly5.1 (Thyl.2) mouse strains were bred

and maintained at the animal care facility at the StanfordSchool of Medicine. All mice were maintained on acidified water(pH 2.5). Mice used as bone marrow donors were 6-12 weeksold, and irradiated recipient mice were more than 8 weeks old.\ Antibodies. The antibodies used in immunofluorescencestaining included 19XE5 (anti-Thyl.1), AL1-4A2 (anti-Ly5.2),2B8 (anti-c-kit), and E13 (anti-Sca-1) (14). For purposes of thispaper, lineage marker antibodies include KT31.1 (anti-CD3),53-7.3 (anti-CD5), 53-6.7 (anti-CD8), Terll9 (anti-erythrocyte-specific antigen), 6B2 (anti-B220), and 8C5 (anti-Gr-1). M1/70(anti-Mac-1) and GK1.5 (anti-CD4) staining of hematopoieticpopulations are characterized separately from the lineagecocktail. Antibodies were usually directly conjugated witheither fluorescein-5-isothiocyanate (Molecular Probes), phy-coerythrin, or allophycocyanin (Cyanotech, Kailua-Kona, HI).

Fetal Liver Preparation and Staining. C57BL/Ka-Thyl.1breeders were put together in the late afternoon and werechecked for vaginal plugs the following morning. The morningon which vaginal plugs were observed was designated 0.5 dayspostcoitum (dpc). Livers were dissected from fetuses, andsingle-cell suspensions were prepared by drawing liver cellsthrough a 25-gauge needle and then expelling them backthrough the needle and through a nylon mesh screen. Cellswere always suspended in Hanks' balanced salt solution(HBSS, without phenol red) (Applied Scientific, San Fran-cisco) containing 2% (vol/vol) calf serum (Irvine Scientific) atpH 7.2. All antibody incubations were for 20 min on ice, priorto diluting the cells and centrifuging through a calf serumcushion. After antibody staining, cells were resuspended inpropidium iodide (PI) at 0.5 j,g/ml.

Progenitor Purification. Whole fetal liver cells were incu-bated with a cocktail of unlabeled lineage marker antibodiesincluding those specific for CD3, CD4, CD5, CD8, Gr-1, andB220. The cells were then washed and incubated with PE-conjugated anti-rat antibody. After washing, unbound anti-ratparatopes were blocked by incubation in 10% (vol/vol) normalrat serum. The cells were then incubated with fluorescein-5-isothiocyanate-conjugated 19XE5, biotin-conjugated Sca-1,phycoerythrin-conjugated Terll9, and allophycocyanin-con-jugated M1/70. Sca-1+ cells were sometimes preenricheckbypositive selection. Cells stained as above were incubated withMACS (Miltenyi Biotec) streptavidin-conjugated magneticbeads at 4°C for 10 min. Then, Texas Red-conjugated avidinwas added for another 10 min. After incubation, the cells werewashed to remove unbound beads and Texas Red-conjugatedavidin. The resuspended cells were passed through a mini-MACS column, and the magnetic fraction was collected as perthe manufacturer's instructions.

Cell Sorting and Analysis. Most analyses and all cell sorts wereperformed on a dual laser FACS (Becton Dickinson), modifiedas described (15). Propidium iodide-positive cells and cells withhigh obtuse scatter or unphysiologically low forward scatter were

Abbreviations: HSC, hematopoietic stem cell(s); dpc, days postcoitum;WBM, whole bone marrow; WBC, white blood cell.*H.D.H. and A.M.W. contributed equally to this work.tTo whom reprint requests should be addressed at: B261 BeckmanCenter, Stanford University Medical Center, Stanford, CA 94305.

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

Proc. Natl. Acad. Sci. USA 92 (1995) 10303

excluded from all sorts and analyses to eliminate dead cells.Reconstitutions in which 10 or fewer sorted cells were injected perrecipient were conducted by sorting each progenitor populationand then resorting in the cloning mode to obtain precise numbersof cells that were essentially pure for the indicated surface markerphenotype. The cells to be injected into each mouse were sortedinto individual wells of a 96-well plate containing 50-100 ,ul ofHBSS with calf serum. Two more cells were sorted into each wellthan were to be injected into each mouse to make up for lossesin the well and syringe (4). The contents of individual wells weredrawn into 0.5-ml insulin syringes and injected into the retroor-bital venous sinuses of mice anesthetized with Metofane (Pitman-Moore, Washington Crossing, NJ).

Reconstitution Analyses. Recipient mice were lethally irra-diated (920 rads) using an x-ray machine operated at 200 kV,delivering 85 rad/min. The radiation was delivered in twodoses, with -3 hr between doses. After irradiation, mice weremaintained on antibiotic water containing neomycin sulfate(1.1 g/liter) and polymixin B sulfate (106 units/liter). Recon-stituted mice were periodically bled via the tail vein to monitorreconstitution by donor-marked progenitors. Red blood cellswere depleted by sedimentation, and the remaining red bloodcells were lysed by a 5-min incubation on ice in 0.15 Mammonium chloride plus 0.01 M potassium bicarbonate. Cellswere then stained with three-color combinations includinganti-Ly5.2 (or anti-Ly5.1 depending on the donor and recipientstrains used) to detect donor cells and either anti-B220 andanti-Mac-1 or anti-CD3 and anti-Gr-1 and analyzed-by FACS.

Cell Cycle Analysis. Fetal liver cells were incubated inHoechst 33342 (Sigma), stem cells were purified by FACS, andHoechst fluorescence was reanalyzed as described (4).

Radioprotection Assay. Fetal liver progenitors were purifiedby sorting and resorting to minimize or eliminate contamina-tion. Recipient mice were irradiated and reconstituted byprogenitors as described above, except that the radioprotectivedoses of syngeneic bone marrow were omitted. The survival ofmice was monitored daily for 30 days.

RESULTS AND DISCUSSIONThy-11iSca-1+Lin-Mac-1+CD4- Fetal Liver Cells Are

Long-Term Reconstituting HSC. We have recently demon-strated that the pool of multipotent progenitors in adult mousebone marrow can be purified and segregated into subpopula-tions of Thy-1OSca-l+Lin-/l1 cells (3, 4). These Mac-1-CD4-,Mac-1'°CD4-, and Mac-1'°CD4'° subpopulations are long-term self-renewing, transiently self-renewing, and a mixture oftransiently and non-self-renewing multipotent progenitors,respectively (ref. 4; unpublished data). These populations werepurified from the fetal liver to determine if populations withthe same cell surface marker phenotypes also contain the hier-archy of primitive progenitors in the fetal liver. Unlike adultbone marrow, distinct Mac-1'° and Mac-lhi populations werenot observed in the fetal liver. As a result, fetal liver popula-tions were characterized as Mac-1+ or Mac-i- (see Fig. 2).Similarly, populations could only be characterized as CD4+ orCD4-. Neither the Thy-1i0Sca-1+Lin-Mac-1-CD4- popula-tion nor the Thy-11OSca-1+Mac-1+CD4+ population fromC57BL-Thyl.1 fetal liver exhibited any detectable progenitoractivity (data not shown). In contrast, the Thy-1iOSca-l+Lin-Mac-l+CD4- population was highly enriched for multipotentprogenitor activity.

Fig. 1 shows the reconstitution profiles of nine lethallyirradiated mice injected with 15 donor-type Thy-1'°Sca-1 +Lin-Mac-1+CD4- cells (hereafter referred to as Lin-Mac-1+CD4- cells) plus a radioprotective dose of 2 x 105 host-typewhole bone marrow (WBM) cells. Five of nine mice werelong-term multilineage reconstituted with donor-type cells.Three mice were not reconstituted by donor-type cells, and onerecipient was transiently reconstituted by donor-type myeloid

and B cells. Thirty-seven weeks after reconstitution, 63% ±23% (mean ± SD) of white blood cells in long-term recon-stituted mice were donor derived.

In the experiment described in Fig. 1, the Lin-Mac-l+CD4progenitor cells were isolated from whole fetal liver by a singleFACS sort. Fig. 2 shows the fluorescence profiles of Lin-Mac-1+CD4- cells upon reanalysis after a single cell sort. Tooptimize purity, further experiments were conducted in whichLin-Mac-1+CD4- cells were purified by two consecutive cellsorts. The results of these experiments, and that shown in Fig.1, are summarized in Table 1. Overall, 77% of reconstitutedmice were reconstituted by long-term self-renewing HSC. Ifonly the experiments with a limit dilution frequency of recon-stituted mice are considered, 67% of Lin-Mac-1+CD4- clonesare long-term reconstituting HSC.

In the experiments in which cells were purified by consecutivesorts, on average, 1 out of 6 ± 3 (95% confidence interval)i.v.-injected cells homed to the bone marrow and read outdetectable progenitor activity. Most i.v.-injected HSC are non-specifically trapped in nonhematopoietic tissues such as the adultliver and lungs (W. H. Fleming, E. Lagasse, I.L.W., unpublisheddata). The homing frequency of multipotent progenitors to thebone marrow after i.v. injection has been estimated at between 1in 5 and 1 in 20 (1, 4, 16, 17); therefore, the reconstitution data

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FIG. 1. Donor-type B, myeloid, and T cells in the peripheral bloodof lethally irradiated mice (a percentage of all white blood cells)injected with 15 donor-type Thy-1iOSca-1+Lin-Mac-1+CD4- cellsplus 200,000 recipient-type WBM cells. Each line represents a singlemouse. Shaded areas represent the range of background signalsobserved in recipients reconstituted by recipient-type WBM cells.Donor-type cells were isolated from the livers of fetuses 13.5 dpc bya single FACS sort. The proportion of unreconstituted mice indicates thatmice were reconstituted at limit dilution, according to Poisson statistics.

Immunology: Morrison et al.

10304 Immunology: Morrison et al.

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are consistent with the Lin-Mac-1lCD4- population being anessentially pure population of multipotent progenitors, mainlyhaving long-term self-renewal potential.Lin-Mac-l+CD4- Population Dynamics. Table 2 shows the

size of fetal liver, the frequency of Lin-Mac-l+CD4- cells inthe fetal liver, and the cell cycle status of Lin-Mac-l+CD4-cells on successive days during midgestation. The fetal liver isthe principal site of hematopoiesis between 12 and 15 dpc (18).During this period, the number of cells in the fetal liver doubleseach day (Table 2; also ref. 12). Yet between days 12 and 14,the frequency of Lin-Mac-l+CD4- cells remains constant;furthermore, a very high proportion ofLin-Mac-1+CD4- cellsappear to be in cycle, as more than a quarter of these cells havegreater than 2n DNA content. While continuous immigrationof Lin-Mac-l+CD4- cells to the liver between 12 and 14 dpccannot be ruled out, the simplest interpretation of these datais that liver Lin-Mac-1lCD4- cells are doubling daily. Thefrequency of fetal liver HSC with greater than 2n DNA contentwas previously estimated at 40% based on Hoechst 33342staining of Thy-1loSca-1+Lin-/l1 cells (19). Fetal liver HSCinclude a much higher frequency of cycling cells than anymultipotent progenitor population in normal adult bone mar-row (4, 19). Only 4% of long-term reconstituting HSC frombone marrow have >2n DNA content (4).By 15.5 dpc, the frequency of Lin-Mac-l+CD4- cells de-

clines significantly (P < 0.05), from 0.044% to 0.015% of fetal

Table 1. Donor cell reconstitution profiles observed uponcompetitive reconstitution of lethally irradiated adult mice byLin-Mac-1+CD4- fetal liver HSC

Dose of Mice Long-term Transient Oligo- Unre-HSC assayed multilineage multilineage potent constituted15 9 5 0 1 310 15 14 0 0 110 18 10 0 2 65 19 5 3 4 7

Lin-Mac-l+CD4- cells were purified by FACS from the livers ofC57BL-Thyl.1 (Ly5.2+) fetuses aged 13-14 dpc. Cells were isolated bya single sort in experiment 1 (15-cell dose), but in all other experi-ments, cells were purified by consecutive sorts. Sorted donor cells wereused to competitively reconstitute lethally irradiated Ly5.1+ adult mice,along with a radioprotective dose (200,000 cells per recipient) of Ly5.1+WBM cells. To be- considered reconstituted, donor-type cells of a parti-cular lineage had to represent >0.3% of white blood cells (WBCs). Micewere considered long-term multineage reconstituted when Ly5.2+ mye-loid, B, and T cells were observed in the blood for at least 16 weeksafter reconstitution. Thirty-two of 34 long-term multilineage reconsti-tuted mice had >20% donor-derived WBCs (all had >10%o). Mice wereconsidered transiently multilineage reconstituted if Ly5.2+ myeloid, B,and T cells were observed in the blood, but Ly5.2+ myeloid cells couldno longer be detected by 16 weeks. Oligopotent reconstitution wascharacterized by transient Ly5.2+ reconstitution of one or two lineages.

liver cells. Since the frequency of dividing cells remains high,this reduction cannot be explained by a decrease in the rate ofdivision of Lin-Mac-1lCD4- cells. Currently, we cannot ruleout the possibilities that most HSC change their surfacemarker phenotype, differentiate, or die between 14.5 and 15.5dpc; however, these possibilities would be unprecedented. At15 dpc the fetal spleen becomes hematopoietic and by 17 dpcthe bone marrow also becomes hematopoietic (18). There is nodirect evidence that other tissues are seeded by HSC from thefetal liver; however, the apparent precipitous decline in fetalliver HSC frequency immediately as hematopoiesis is shiftingto other tissues is compelling evidence that large numbers ofHSC may be emigrating from the fetal liver to seed othertissues. Indeed, this process may be analogous to the observedmobilization of HSC to blood and spleen in adults (20-22).Lin-Mac-l+CD4- Cells Are the Only Long-Term Recon-

stituting Fetal Liver HSC. Lethally irradiated adult mice werecompetitively reconstituted with serial dilutions of whole fetalliver cells to determine the frequency of long-term reconsti-tuting HSC in the fetal liver. The results are presented in Table3. The data in Table 3 can be described by a regressionequation computed using SYSTAT software (Systat, Evanston,IL) according to the model described by Smith et al. (1):

log(PO) = -2.7 x 10-5(N) - 0.009,

where Po = the proportion of mice that are not long-termreconstituted by donor-type cells at a particular dose of wholefetal liver cells andN = the number of donor-type whole fetalliver cells injected per mouse (squared multiple R = 0.983;ANOVA P = 0.009).

Poisson statistics predict that mice are reconstituted onaverage by a single long-term reconstituting HSC when 63% ofmice assayed are long-term reconstituted by donor-type cells(Po = 0.37). Based on the above regression equation, onaverage, a single long-term reconstituting HSC homes to a

Table 2. Fetal liver size and the frequency and cell cycle status ofLin-Mac-l+CD4- cells at sequential days during midgestation

12.5 13.5 14.5 15.5Parameter dpc dpc dpc dpc

Cells per fetal liverx 10-6* 3.1±2 6.4±3 11.6±6 29±7

Lin-Mac-1+CD4-frequency,t % 0.040 0.038 0.044 ± 0.014 0.015 ± 0.008

Cell cycle status, %in S + G2 + M 25 31 27 26

*Number of cells per fetal liver (mean ± SD). Data are based on threeto five pooled samples of 4-19 fetal livers each.

tPopulation frequency (mean ± SD) is a mean of two or fivedeterminations.

Proc. Natl. Acad. Sci. USA 92 (1995)

Proc. Natl. Acad. Sci. USA 92 (1995) 10305

Table 3. Limiting dilution reconstitution of congenic adult micewith 14 dpc whole fetal liver to determine the frequency oflong-term reconstituting HSC

Dose of Long-term Transientwhole fetal Mice multi- multi- Lymphoid Unre-liver cells assayed lineage lineage only constituted

200,000 3 3 0 0 0100,000 5 5 0 0 050,000 10 10 0 0 025,000 10 8 0 1 112,500 10 5 2 2 16,000 9 4 0 4 1

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Whole fetal liver cells were obtained from 14 dpc C57BL-Thyl.1(Ly5.1+) fetuses. Serial dilutions of these cells were used to compet-itively reconstitute lethally irradiated Ly5.2+ adult mice, along with aradioprotective dose (200,000 cells per recipient) of Ly5.2+ WBMcells. Reconstitution profiles were as described in the legend to Table1. All long-term multilineage reconstituted mice had >20% donor-derived WBCs.

hematopoietic tissue and reads out progenitor activity when15,700 whole fetal liver cells are injected. Assuming stem cellshome to the bone marrow and read out progenitor activity ata frequency of one in six cells, as observed for the Lin-Mac-1+CD4- population, the frequency of long-term reconstitutingHSC in the day 14 fetal liver is 1 in 2600 cells, or 0.038%.Unfortunately, the standard error associated with this estimatecannot be estimated due to the nonuniform error distributionof the data. The observed frequency of Lin-Mac-l+CD4-cells in the day 14.5 fetal liver is 0.044% on average. Thus thefrequency of long-term reconstituting HSC activity in thewhole fetal liver is consistent with the Lin-Mac-1+CD4-population containing all of the long-term reconstituting HSC.The frequency of transiently reconstituting HSC cannot be

accurately estimated among whole fetal liver cells or in theLin-Mac-l+CD4- population because long-term reconstitu-tion with donor-type HSC would mask reconstitution bytransiently reconstituting HSC. Nonetheless, fewer transientthan long-term multipotent progenitors are observed in thelimit dilution doses of both the Lin-Mac-l+CD4- populationand in the whole fetal liver. This contrasts with the 4:1 ratio oftransient to long-term HSC in the adult bone marrow (4, 16).The Lin-Mac-l+CD4- population may contain all of themultipotent progenitors observed in the fetal liver includingtransient and long-term progenitors.The distribution of long-term progenitor activity was also

examined by separating whole fetal liver cells into positive andnegative fractions for particular antigens, as has been donewith other tissues or markers (5, 13). Then the number ofantigen-positive or -negative cells equivalent to the number ofcells of that population contained in 50,000-200,000 wholefetal liver cells was used to competitively reconstitute lethallyirradiated adult recipients. As shown in Table 3, doses of 5 x104 to 2 x 105 whole fetal liver cells were always sufficient tolong-term multilineage reconstitute all recipients. Thus HSCsufficient to long-term reconstitute all recipients should beconcentrated in the fractions that reflect antigen expression byHSC. The distinction between fractions should be very clearwhen HSC express an antigen at high levels and are easilyseparated from negative cells. The distinction would be ex-pected to be more qualitative when HSC are negative orexpress low levels of an antigen, where the distinction betweenpositive and negative cells is fine enough that when sortinglarge numbers of cells, some antigenlo cells would contaminatethe antigen-negative fraction, and vice versa.The results of this experiment are shown in Table 4. All

long-term reconstituting HSC from the fetal liver appear to bec-kit+ and Mac-1+, and the data strongly suggest that all HSC

Table 4. Characterization of markers expressed by long-termreconstituting HSC by separating whole fetal liver into positiveand negative fractions for particular antigens

Fraction of mice long-term multilineage

Antigen used to reconstituted with donor cellsseparate donor cells Antigen negative Antigen positive

Sca-1 1/5 5/5Thy-1.1 4/13 11/12Mac-1 0/7 7/7CD4 9/9 2/3Lineage 10/10 4/13c-kit 0/5 5/5

Whole fetal liver cells from C57BL-Thyl.1 fetuses 12.5-14 dpc wereseparated by FACS into positive and negative fractions based on anti-gen expression. A dose of donor-type separated cells equivalent to thenumber of cells of that population contained in 50,000-200,000 wholefetal liver cells was injected along with a radioprotective dose of recip-ient-type WBM cells into adult recipients. Long-term multilineage recon-stituted mice were as defined in the legend to Table 1. All but one long-term multilineage reconstituted mouse had >20% donor-derived WBCs.

are Sca-1+. As predicted, the results are more qualitative withthe Thy-1, Lin, and CD4 antigens. The data demonstrate thatmost HSC are Thy-110, Lin- (referring to antigens other thanMac-1 and CD4), and CD4- but cannot rule out minorpopulations of HSC with the opposite antigen phenotype.Long-term reconstituting activity was observed in the CD4+fraction, yet when Thy-11OSca-lMac-1+CD4± cells were as-sayed for progenitor activity, none was observed. Some low-level nonspecific staining is a general problem with GK1.5 inour hands. The results of this experiment are consistent withthe Lin-Mac-1+CD4- population containing all of the long-term reconstituting HSC in the fetal liver.

Fetal Liver Versus Bone Marrow HSC. Limit dilution dosesof purified long-term reconstituting HSC (donor type) fromfetal liver or bone marrow (4) were competed against 2 x 105WBM cells (recipient type) for the reconstitution of lethallyirradiated adult recipients. In each case, the representation ofdonor-derived WBCs in long-term multilineage reconstitutedmice was observed over time. Three independent experimentswere conducted with bone marrow-derived HSC, and threewere performed with fetal liver-derived HSC. The results arepresented in Fig. 3. Fetal liver HSC tended to repopulate to agreater degree than bone marrow-derived HSC, especiallybetween 4 and 16 weeks after reconstitution (see also ref. 23).If fetal liver HSC reconstitution data are pooled and comparedto pooled bone marrow HSC reconstitution data, fetal livercells reconstitute to a significantly greater degree for at leastthe first 16 weeks after reconstitution (P < 0.001). If experi-ments are considered individually, one of three experimentsusing fetal liver HSC yielded significantly higher levels ofreconstitution (P < 0.05) than each of the bone marrow HSCreconstitution experiments. Despite the apparent increasedcompetitiveness, the radioprotective capacity of fetal liverHSC was indistinguishable from that of adult bone marrowmultipotent progenitors (4), as 80, 40, or 20 cells radiopro-tected 90%, 50%, or 10% of recipients, respectively (data notshown). Whole fetal liver cells were previously observed torepopulate irradiated adult mice to a greater extent than anequal number of adult bone marrow cells (6, 13). In additionto being more competitive, long-term reconstituting HSC arealmost 7 times more frequent in the fetal liver (up to 15 dpc)than in the adult bone marrow.There are now several examples of phenotypic and func-

tional changes documented in HSC. For example, human HSCfrom adult bone marrow were HLA-DR- (24), whereas thosefrom the fetal bone marrow (25) or cord blood (26) wereHLA-DR+. Mouse fetal liver HSC are AA4.1+ (8, 13), butadult bone marrow HSC are not (27). Functionally, mouse

Immunology: Morrison et al.

10306 Immunology: Morrison et al.

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FIG. 3. The frequency of donor-type white blood cells (as apercentage of all white blood cells) in the peripheral blood of micereconstituted by limit dilution doses of purified long-term reconstitutingHSC. Means of all long-term multilineage reconstituted (LTMR) mice ineach experiment are presented. Fifteen fetal liver (FL) HSC reconstituted6 of 9 mice, of which 5 were LTMR; 10 fetal liver HSC reconstituted 12of 18 mice, of which 10 were LTMR; and 5 fetal liver HSC reconstituted12 of 19 mice, of which 5 were LTMR. Five bone marrow (BM) HSCreconstituted 4 of 14 mice, of which 3 were LTMR; 10 bone marrow HSCreconstituted 4 of 10 mice, of which 3 were LTMR; and 10 bone marrowHSC reconstituted 15 of 20 mice, of which 12 were LTMR.

fetal HSC can respond to the fetal thymic environment byproducing V.3' and Vy4+ T cells, whereas adult HSC cannot(11). Adult HSC are deficient relative to fetal HSC in theirability to reconstitute Bla lymphocytes (28, 29). Lansdorp etal. (30) have shown that candidate human HSC purified frombone marrow, cord blood, or fetal liver exhibit differences atthe population level in proliferative potential in vitro.We now demonstrate phenotypic and clonal functional dif-

ferences between fetal liver and bone marrow HSC in vivo. Inthe fetal liver of our mice, all long-term reconstituting HSCappear to be Mac-1+. In adult bone marrow, long-term recon-stituting HSC are Mac-i-, whereas transiently reconstitutingHSC are Mac-1+ (4). The consistent expression of Mac-1 bymultipotent progenitors in different tissues and at differenttimes during ontogeny suggests that Mac-1 may play a func-tional role on HSC. Mac-1, a heterodimer ofCD1 lb and CD18,is a member of the integrin family. CD18 and CD1lb are adhe-sion molecules (31-33), and CD18 may play a role in the inter-action of multipotent progenitors with human bone marrowstroma (34, 35). Perhaps the differences in Mac-1 expressionby HSC in the fetal liver and the bone marrow relate to changesin environmental signals. Mac-i-expressing multipotent pro-genitors tend to be proliferative, whereas Mac-i- HSC tend tobe quiescent (4). These observations hint that Mac-1 expres-sion could play a role in the transmission of proliferation sig-nals to multipotent progenitors. These findings amplify the factthat correlations between cell function and surface marker ex-

pression that are found to apply in one tissue cannot be pre-sumed to apply in other tissues or under other conditions (17, 36).

We thank L. Jerabek for laboratory management, V. Braunstein forantibody preparation, and T. Knaak for operation of the FACSmachines used in this study. Thanks also to L. Hidalgo and R. Salazarfor animal care and to A. Schlagetter for reading the manuscript.S.J.M. is a Howard Hughes Medical Institute Predoctoral Fellow. Thiswork was supported by National Cancer Institute Grant CA42551.

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2. Visser, J. W., Bauman, J. G. J., Mulder, A. H., Eliason, J. F. & deLeeuw, A. M. (1984) J. Exp. Med. 159, 1576-1590.

3. Spangrude, G. J., Heimfeld, S. & Weissman, I. L. (1988) Science241, 58.

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