Proc. Nati. Acad. Sci. USAVol. 89, pp. 3790-3794, May 1992Cell Biology

Purification and characterization of retrovirally transducedhematopoietic stem cellsLISA M. SPAIN* AND RICHARD C. MULLIGANtWhitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142; and Department of Biology, Massachusetts Institute ofTechnology, Cambridge, MA 02139

Communicated by Stuart Orkin, December 3, 1991 (receivedfor review October 16, 1991)

ABSTRACT We have developed a method for the purifl-cation of retrovirally transduced hematopoietic stem cells,based on a modification of the stem cell purification protocolsdeveloped by Spangrude et al. [Spangrude, G., Heimfeld, S. &Weissman, I. (1988) Science 241, 58-64] and Spangrude andScollay [Spangrude, G. & Scollay, R. (1990) Exp. Hematol. 18,920-926], that depends upon the use of bone marrow cellsisolated from 5-fluorouracil-treated mice that have been sub-sequently cocultivated with recombinant retrovirus-producingcell lines. We found that purified cell populations bearing a Sca1+ Lin- Thy 1- surface phenotype represent a 50-100% purepopulation of spleen colony-forming cells (CFU-S day 12).Animals injected with 300 or more purified cells were consis-tently radioprotected and reconstituted in multiple lineageswith donor cells. Sca 1+ Linf Thy 1- CFU-S day 12 stem cellswere shown to be efficiently (100%) transduced by the recom-binant retroviruses used in the study. Gene transfer intolong-term reconstituting stem cells, as evidenced by Southernblot analysis of mature hematopoietic cell types 3 months aftertransplantation, was observed only in recipients injected withlarge numbers (-4000-5000) of the purified cells. The devel-opment of methods for purifying retrovirally transduced stemcells should prove extremely useful for various studies in whichit is of interest to characterize the activity of a specific geneproduct (e.g., growth factor, receptor, oncogene) specifically inprimitive hematopoietic cell types.

In the past several years, a number of methods for thepurification of hematopoietic stem cells have been reported(1-15). In general, these methods depend upon the fraction-ation of bone marrow or fetal liver cells on the basis of eithertheir size and density, their ability to bind lectins and vitaldyes, or their expression of various cell surface antigens.Though all of these studies have provided important insightsregarding the functional properties of hematopoietic stemcells, they have also raised important questions about thecomplexity of the stem cell pool, the role of different popu-lations of cells in normal hematopoiesis and hematopoiesisfollowing bone marrow transplantation, and the validity ofdifferent in vitro and in vivo assays for stem cell function.

Retroviral marking studies have also raised questionsregarding the heterogeneity of the stem cell pool. Althoughwe and others have provided convincing evidence that cellscapable of the long-term reconstitution of lethally irradiatedrecipients can be transduced by recombinant retroviruses(16-22), it is clear that a rather complex population ofhematopoietic cells possessing varying self-renewal and dif-ferentiation properties exists, as evidenced by the variable,and in some cases transient, contributions oftransduced cellsto hematopoiesis that have been observed in those experi-ments.

Because of our efforts aimed at developing efficient meth-ods for transferring genes into hematopoietic cells in vivo (16,18, 23, 24), we are particularly interested in characterizingstem cell populations able to permanently reconstitute le-thally irradiated recipients and in determining the uniqueproperties, if any, of stem cells susceptible to retrovirusinfection. Toward these ends, we describe here the devel-opment of methods for the purification of retrovirally trans-duced hematopoietic stem cells and the characterization ofthe purified cells.

MATERIALS AND METHODSRetroviral Infection of Bone Marrow. Murine bone marrow

cells were isolated and cocultivated with virus-producingcells as described (16). The retroviral producing cell line F9,used in these studies, produced ==1-5 x 106 colony-formingunits (CFU)/ml (P. Robbins, P. Lehn, and R.C.M., unpub-lished data).Stem Cell Purification and Hematopoietic Reconstitution.

Infected, Ficoll-purified marrow cells were incubated in acocktail of rat monoclonal antibodies (kindly provided by I.Weissman and S. Heimfeld, Stanford) against the antigensMac 1 (M1/70), Gr 1 (RB6-8C5), B220 (RA3-6B2), Lyt 2(53.6.7), L3T4 (GK1.5), Ly 1 (53.7.3), _108 cells per ml inHepes-buffered saline plus 2% fetal calfserum (HBS/FCS) asdescribed (1). After washing by centrifugation through a100% serum cushion, the cells were incubated with goatanti-rat allophycocyanin (Caltag), washed again as before,and blocked by incubation for 10 min in 1:3 dilution of ratserum (Cappel Laboratories). Phycoerythrin-conjugated an-ti-Thy 1 (Caltag) and biotinylated anti-Sca 1 (provided by I.Weissman, Stanford University) were added to the blockingmixture, incubated for 20 min on ice, and washed. The cellswere next incubated with anti-biotin fluorescein isothiocya-nate (Sigma), washed, and resuspended in HBS/FGS with 2,g of propidium iodide per ml (Sigma). The cells were thensubjected to four-color analysis and sorting on a FACStarplus instrument (Becton Dickinson). C57BL/6 recipientswere irradiated and transplanted with cells as described (16).

Southern Blot Analysis. DNA was isolated from specificcell populations prepared from transplant recipients anddigested, electrophoresed, immobilized on filters, and hy-bridized as described (16). Detection of donor cell DNAsequences, neomycin (neo)-specific sequences, and interleu-kin 3 (IL-3)-specific sequences was accomplished with spe-cific 32P-labeled probes as described (16).

Abbreviations: 5FU, 5-fluorouracil; CFU-S, spleen colony-formingunit(s); neo, neomycin; IL-3, interleukin 3; FACS, fluorescence-activated cell sorting.*Present address: Department of Cellular and Developmental Biol-ogy, Harvard University Biological Laboratories, 16 Divinity Av-enue, Cambridge, MA 02138.tTo whom reprint requests should be addressed.

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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"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 89 (1992) 3791

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FIG. 1. 5FU treatment increases the percentage of Sca 1+ Lin- Thy 1- cells in mouse bone marrow. Shown are dot plots of 10,000 untreated(A-C) or 5FU-treated (D-F) bone marrow cells analyzed for Sca 1, Lin (Mac 1, Gr 1, B220, Ly 1), or Thy 1 antigen expression as indicated.The boxes shown in A and D indicate the typical window used for sorting of Sca 1+ Lin- cells. B and E show the relative expression of Thy1 on cells gated for Sca 1' Lin- phenotype as indicated in A and D. 5FU treatment also increases the percentage of Thy lin' Lin- marrow;compare the percentage of cells in the lower righthand quadrant indicated in C for untreated and F for 5FU-treated bone marrow.

RESULTS

In light of the findings by our laboratory and others that5-fluorouracil (5FU) pretreatment of donor mice appears toincrease the proportion ofreconstituting stem cells infectable byretrovirus vectors (refs. 16, 17, 19, 23, 24; unpublished results),we chose to develop a method for isolating stem cells compat-ible with our standard infection protocol. The starting point forour studies was to determine whether 5FU treatment andsubsequent cocultivation ofbone marrow cells with retrovirus-producing cell lines had any effects on expression of the cellsurface markers employed by Spangrude et al. (1) for thepurification of stem cells. Fig. 1 shows a two-color fluores-cence-activated cell sorting (FACS) analysis ofnormal or 6-dayposttreatment 5FU marrow cells (that were previously cocul-tivated with virus-producing cell lines) for the expression ofeither Lin (Mac-1, B220, Gr-1, Ly 2, L3T4, Ly 1) and Sca 1 (Ly6A/E) (Fig. 1A) or Lin and Thy 1 (Fig. 1C) antigens. The resultsindicate that 5FU treatment significantly increased the propor-tion of Lin- cells in general and specifically increased theproportion of Lin- Sca 1+ and Lin- Thy 1- cells. Since theabsolute level of Thy 1 expression has also been shown bySpangrude et a!. to be an important indicator of the stem cellsurface phenotype, we also examined the Sca 1+ Lin- fractionof normal and 5FU-treated marrow cells for the level of Thy 1

expression. One broad peak of Thy 1 expression (termedintermediate, or Thy lt), representing about halfof the Sca 1+Lin- cells, was observed whether or not5FU marrow was used(Fig. 1B). No discernible fraction of cells displayed lowamounts of Thy 1 (Thyow), as was described by Spangrude etal. (1) in their studies. Although the reason for this discrepancyis unclear, it may be that 5FU treatment and/or cocultivation ofcells alters their Thy 1 phenotype. Alternatively, our FACSanalyses may not be sensitive enough to permit detection ofThy1Iw cells.The day 12 spleen colony formation assay provides a

reliable estimate of self-renewing myeloerythroid stem cellnumbers (25). Therefore, we next examined the effects of5FU on the absolute number and proportion ofCFU-S day 12cells in unfractionated and fractionated cell populations (Ta-ble 1). As reported before (26), 5FU treatment of micesignificantly reduced the overall cellularity of the marrow.The absolute number of CFU-S day 12 cells, however,decreased at day 2 and then rebounded to levels higher thannormal marrow at day 6. Accordingly, the proportion ofCFU-S day 12 cells present in day 6 posttreatment 5FUmarrow increased 15- to 20-fold over that found in normalmarrow. Purification of Sca 1+ Lin- cells from normalmarrow resulted in close to a 100-fold enrichment of CFU-S

Table 1. 5FU increases the frequency and number of CFU-S day 12 stem cells in Sca 1+ Lin- fractions

Day 12 spleen colonies per 100 cells injected

Time after Cells per Sca 1*Lin-5FU treatment, femur plus CFU-S-12 cells per Total* Sca 1+ Lin-*__Thy 1-*

days tibia femur plus tibia No. n No. n No. n

Untreated 7.5 x 106 1350 0.018 ± 0.008 7 1.37 ± 0.5 15 2.0 ± 0.5 22 2.5 x 106 125 0.005 ± 0.003 4 1.16 ± 1.0 5 ND6 0.85 x 106 2550 0.300 ± 0.1 4 5.5 ± 0.3 3 7.6 ± 2.6 4

CFU-S day 12 cells were assayed using three or more recipient mice per independent determination. The number of colonies obtained varied'20%o among spleens within experiments. The values of numbers of CFU-S day 12 cells are averages of two or more independent experiments.ND, not determined.*Values are expressed as mean ± SD; n indicates number of experiments.

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Table 2. Proportion of total bone marrow-derived CFU-S day 12cells found in the Sca 1+ Lin- Thy 1- fraction

No. of spleen coloniesper 100 cells injected

Exp. Unfractionated Sca 1+ Lin- Thy 1- Fraction* %tA 0.3 6.0 0.054 108B 0.25 4.5 0.042 76C 0.5 5.5 0.09 99

The CFU-S day 12 content of cell populations was determinedthrough the transplantation of cells into at least three recipients. Thenumber of colonies obtained varied <20%o from animal to animal. A,B, and C represent three independent experiments.*Fraction of total bone marrow cells that are Sca 1+ Lin- Thy 1-(determined by FACS analysis).t% total bone marrow-derived CFU-S day 12 cells found in Sca 1+Lin- Thy 1- fraction.

day 12 cells, as reported (2), whereas in the case of 5FUmarrow, which is already enriched, Sca 1+ Lin- purificationled to an additional 10- to 20-fold enrichment. Interestingly,the Sca 1+ Lin- fraction isolated from 5FU marrow containedup to four times as many CFU-S day 12 cells as the corre-sponding fraction from normal marrow. Further purificationof Sca 1+ Lin- Thy 1- cells led to a slightly higher enrichmentof CFU-S day 12 activity. When allowances are made forspleen seeding efficiency (4, 6), the Sca 1+ Lin- Thy 1-fraction was a 50-100% pure population of CFU-S day 12cells. In terms of the overall recovery ofCFU-S day 12 cells,we found that 94% ± 12% of the CFU-S day 12 cells foundin unfractionated 5FU-treated marrow could be accountedfor by assay of the Sca 1+ Lin- Thy 1- fraction (Table 2).To determine the transduction efficiency ofCFU-S day 12

cells generated from Sca 1+ Lin- Thy 1- cells, DNA from thespleens of mice transplanted 12 days earlier with Sca 1+ Lin-Thy 1- cells isolated from infected bone marrow cells (eithernormal or 5FU treated) was examined for proviral DNAsequences. In each case, the number of cells transplantedwas enough to form :60 colonies per spleen. By analyzingspleens confluent with colonies, we were able to assayretroviral infection of a larger number of CFU-S day 12 cellsthan would be possible by assaying each colony individually.As shown in Fig. 2, all DNA samples from the animalstransplanted with Sca 1+ Lin- Thy 1- cells isolated from 5FUmarrow show evidence of efficient transduction (at least onecopy per cell). In contrast, spleenDNA samples from animalsengrafted with Sca 1+ Lin- Thy 1- cells from normal marrowshowed only a low level of proviral sequences.

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FIG. 2. 5FU increases the retroviral infectivity of Sca 1+ Lin-Thy 1- CFU-S day 12 stem cells. One thousand purified Sca 11 Lin-Thy 1- cells from 5FU-treated mice and 3000 Sca 1+ Lin- Thy 1-cells from normal mice were injected into lethally irradiated animalsto generate spleens confluent with colonies. The confluent spleenswere harvested at 12 days, and chromosomal DNA was prepared anddigested with Kpn I, which cleaves within the long terminal repeatand therefore excises an identical proviral DNA-containing DNAfragment, regardless of the proviral site of integration. The digestedDNA was analyzed by the method of Southern and probed forproviral sequences as described (16). The colonies were derived fromSca 1+ Lin- Thy 1- cells isolated from normal (lanes 1-4) or day 6posttreatment 5FU (lanes 5-10) marrow.

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FIG. 3. Extent and distribution ofdonor reconstitution ofanimalstransplanted with 30-100 Sca 1+ Lin- Thy 1- stem cells. Animal 1Areceived 30 cells; animals 2A, 2B, and 2C received 100 cells each.Cells and tissues from animals sacrificed at 12 weeks were fraction-ated, and their DNAs were cut with EcoRI, blotted, and probed withZfy sequence. The bands representing Zfy, Zfx, and Zfa are indicatedand the faint band above Zfa is a Zfx-specific partial restrictionfragment (27). The intensity of the Zfa signal serves as an internalcontrol for DNA loading (see controls). Lane designations: s, spleen;t, thymus; m, marrow; 1, lymph node; p, peritoneal exudate cell; Bs,spleen-derived B cells; Bm, marrow-derived B cells; Ms, spleen-derived macrophages; Mm, marrow-derived macrophages. (A) An-imals 1A and 2A. (B) Animals 2B and 2C.

Finally, we examined the ability of the purified cells toradioprotect lethally irradiated recipients and to contribute tohematopoiesis in mice engrafted for long periods of time. Todetermine whether radioprotected recipients were donor orhost reconstituted, male donors were used and DNA isolatedfrom fractionated cell populations from transplant recipientswas analyzed for the presence of Y chromosome-specificsequences (27). In all of the long-term reconstitution exper-iments described, animals were analyzed 12 weeks aftertransplantation. Animals receiving <300 purified Sca 1+ Lin-Thy 1- cells were variably protected from lethal irradiationand showed variable donor reconstitution. For example, twoof four animals transplanted with 30 cells survived 12 weeks,although one of the animals was extremely anemic with ahypocellular marrow. The other mouse, animal 1A, showeddonor reconstitution only in spleen, lymph node, peritonealexudate, and spleen-derived B-cell fractions (Fig. 3). Three offive animals transplanted with 100 cells survived. One ofthese animals, 2B, showed predominant donor reconstitu-tion, whereas the others, 2A and 2C, showed donor contri-butions only in the spleen, thymocyte, lymph node, perito-neal exudate, and spleen-derived B-cell fractions (Fig. 3). Ofanimals receiving >300 purified cells, two of four animalsgiven 300 cells, five of five animals given 1000 cells, and fourof four animals given 3000 cells survived. Two animals foreach of these latter groups were analyzed for donor recon-stitution and all were shown to be engrafted predominantlywith donor cells (Fig. 4). In two other experiments, alsoanalyzed at 12 weeks after transplantation, three of threeanimals given 4500 Sca 1+ Lin- Thy 1- cells and three ofthree animals given 5000 Sca 1+ Lin- cells survived and weredonor reconstituted (Fig. 5A).

Surprisingly, in spite of the apparent efficient transductionof Sca 1+ Lin- Thy 1- cells, as evidenced by analysis ofCFU-S day 12 cells (Fig. 2), only one transplant recipient

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FIG. 4. Extent and distribution ofdonor reconstitutionand retroviral infection of animals transplanted with 300-3000 Sca 1+ Lin- Thy 1- stem cells. Animals 3A and 3Bwere transplanted with 300 cells, animals 4A and 4Breceived 1000 cells, and animals 5A and 5B received 3000cells. Each DNA sample was digested with Kpn I, probedfor neo and Y sequences (Upper), and stripped and re-probed for IL-3 sequences as a control (Lower). Ten, 5,and 2.5 ,mg of single-copy neo virus-infected male DNAwas included as a control (far right). See Fig. 3 legend forlane designations.

engrafted with <3000 cells had proviral sequences present inits tissue DNA (Fig. 4). In this one case, 3A, a low level ofprovirus was observed in the lymph node DNA and perhapsin the peritoneal DNA fraction. In one experiment, in whichthe number ofinjected cells was varied from 300 to 3000, onlyone of the two mice transplanted with 3000 cells and exam-ined for proviral sequence, SB, showed evidence of multi-lineage gene transfer. In two other experiments, in whichthree animals were transplanted with 4500 Sca 1+ Lin- Thy1- cells and three animals were transplanted with 5000 Sca 1+Lin- cells, five of the six animals showed evidence ofengraftment by infected stem cells (Fig. 5). In these animals,the proviral copy number in different cell populations rangedfrom 0.1 to 0.25 copy per cell. To identify the integration sites

A

unique to each infection event, theDNA from isolated tissuesas well as from expanded lymphoid and myeloid cell typeswas digested with restriction enzymes that cut once outsideof the provirus (16). In every animal at least one proviralinitegration common to myeloid and lymphoid lineages wasdetected by Southern analysis. Bands that are present inevery lineage are marked by arrowheads in Fig. 5. In addi-tion, in all five animals, apparently myeloid or lymphoidrestricted clones were observed.

DISCUSSIONPerhaps the most important practical aspect of this work isthe successful development of a stem cell purification pro-

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FIG. 5. Proviral DNA copy number in cells for animals reconstituted with Sca 1+ Lin- or Sca 1+ Lin- Thy 1- cells. DNA from spleen (s),thymus (t), marrow (m), and lymph node (1) was probed for retrovirus-derived neo gene sequences. Animals were repopulated with 4000-5000Sca 1+ Lin- Thy 1- stem cells (animals 6A, 6B, 6C) or Sca 1+ Lin- stem cells (animals 7A, 7B, 7C). B, B cells; M, macrophages; T, T cells.(A) Retrovirus-infected stem cells contribute to long-term hematopoiesis. Shown are DNAs from spleen, thymus, marrow, and lymph nodedigested with Kpn I, which cuts within the long terminal repeats, and probed for retrovirus-derived neo sequences. The level of contributionby infected cells is less than single copy, by comparison to the single copy control (lane c). The blots were reprobed for IL-3 and Y sequences,as indicated, to control for DNA loading and the extent of donor repopulation, respectively. (B) Single clones give rise to myeloid and lymphoidlineages. DNAs from tissues and individual lineages were probed for retrovirus-derived neo sequences and were digested with Bgi 11 (B) orEcoRI(E). These enzymes cut within the provirus only once, and thus fragment size is dependent on flanking restriction sites and is specific for eachintegration. In every animal at least one band (marked with arrowheads) is common to all lineages.

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tocol compatible with protocols for high-efficiency retrovi-rus-mediated gene transfer. Our results indicate that thispurification method is among the most efficient reported andmay lead to the isolation of a homogeneous population ofCFU-S day 12 cells, if previously reported estimates ofspleen seeding efficiency are accurate. Furthermore, we havefound that our purification method is also among the mosteffective, resulting in the recovery of most, if not all, CFU-Sday 12 stem cells found in 5FU-treated bone marrow. Theability to purify previously transduced stem cells shouldmake possible various experiments in which it is of interestto assess the activity ofgene products (e.g., protooncogenes,activated oncogenes, growth factor receptors) specifically inprimitive hematopoietic cell types such as CFU-S day 12cells.One straightforward conclusion from our studies is that

individual stem cells enriched in the Sca 1+ Lin- Thy 1-fraction are indeed multipotent and capable of the long-termreconstitution of lethally irradiated recipients. Such resultsare consistent with recent studies by Weissman and co-workers (28) involving the transplantation of single stem cellsand previous studies by Spangrude et al. (1) involving thetransplantation of small amounts (for example, 30 cells) ofpurified cells. Although we cannot directly calculate from ourresults the proportion of cells in the Sca 1+ Lin- Thy 1-fraction capable of long-term reconstitution, we estimate thatit is comparable to that achieved by other purification meth-ods (1-6, 8), since as few as 100 cells were able to reconstitutelethally irradiated mice, and as few as 300 cells did soconsistently.A second important finding of the current studies was that

CFU-S day 12 cells and reconstituting stem cells showedstrikingly different levels of infectivity by recombinant ret-roviruses. CFU-S day 12 cells were efficiently transduced,whereas long-term reconstituting stem cells were less effi-ciently infected, on average. These results provide furtherevidence that spleen colony-forming cells are at least partiallydistinct from long-term reconstituting cells. An increasingbody of evidence in support of this idea has recently emergedfrom conventional cell fractionation experiments (7, 14, 15).The finding that proviral sequences were detectable in the

myeloid and lymphoid cells of recipients transplanted onlywith relatively large numbers of purified cells is surprising inlight of our previous studies, which indicated that a largeproportion of cells of the hematopoietic system in bonemarrow transplant recipients was retrovirally marked after invitro cocultivation of unfractionated bone marrow cells withvirus-producing cell lines. The inability to detect efficientgene transfer when low numbers of purified cells weretransplanted (despite efficient donor repopulation) could bedue to the selective loss of infected stem cells during thecocultivation or purification procedures. However, our datasuggest that when large numbers of purified cells are given,infected stem cells contribute to hematopoiesis to a greaterextent than would be predicted by their low frequency. Oneexplanation is that retrovirus-infected stem cells are prefer-entially used for engraftment. This model would predict thata large proportion of mature hematolymphoid cells could beretrovirally marked even if only a small proportion of the

stem cell pool is infected. Direct measurement of retroviralinfection ofthe purified stem cell fraction may help to resolvethis issue. Clearly, understanding the relationship betweenretroviral infectivity and the probability a stem cell is utilizedfor short- or long-term hematolymphoid reconstitution hasimportant implications for ongoing studies aimed at improv-ing the efficiency of gene transfer to hematopoietic stemcells.

We are indebted to Shelly Heimfeld and Irv Weissman for sharingreagents with us and for helpful advice, to Stewart Connor for helpingwith the FACS analysis and sorting at the M.I.T. Flow CytometryLaboratory, to Paul Robbins for providing retroviral constructs andproducers essential to this work, and to the members of our labo-ratory for useful advice and criticism. This work was supported byNational Institutes of Health Grant R01 HL37569.

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Proc. Natl. Acad. Sci. USA 89 (1992)