blood mag article

1997 90: 85-96

and Gordon Keller Christopher J. Hogan, Elizabeth J. Shpall, Oren McNulty, Ian McNiece, John E. Dick, Leonard D. Shultz

Mice scid/scidUmbilical Cord Blood in NOD/LtSz--Enriched Cells From+Engraftment and Development of Human CD34

http://bloodjournal.hematologylibrary.org/cgi/content/full/90/1/85Updated information and services can be found at:

(2766 articles)Hematopoiesis and Stem Cells collections: BloodArticles on similar topics may be found in the following

http://bloodjournal.hematologylibrary.org/misc/rights.dtl#repub_requestsInformation about reproducing this article in parts or in its entirety may be found online at:

http://bloodjournal.hematologylibrary.org/misc/rights.dtl#reprintsInformation about ordering reprints may be found online at:

http://bloodjournal.hematologylibrary.org/subscriptions/index.dtlInformation about subscriptions and ASH membership may be found online at:

. Hematology; all rights reservedCopyright 2011 by The American Society of 20036.the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by

For personal use only. by on January 13, 2011. www.bloodjournal.orgFrom

http://bloodjournal.hematologylibrary.org/cgi/content/full/90/1/85

http://bloodjournal.hematologylibrary.org/cgi/collection/hematopoiesis_and_stem_cells

http://bloodjournal.hematologylibrary.org/misc/rights.dtl#repub_requests

http://bloodjournal.hematologylibrary.org/misc/rights.dtl#reprints

http://bloodjournal.hematologylibrary.org/subscriptions/index.dtl

http://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtl

http://bloodjournal.hematologylibrary.org


Engraftment and Development of Human CD34"-Enriched Cells FromUmbilical Cord Blood in NOD/LtSz-scid/scid Mice

By Christopher J. Hogan, Elizabeth J. Shpall, Oren McNulty, Ian McNiece, John E. Dick, Leonard D. Shultz,and Gordon Keller

Understanding the repopulating characteristics of human sessing more mature cell surface markers, comprise thehematopoietic stem/progenitor cell fractions is crucial for human component of mouse spleen and peripheral blood,predicting their performance after transplant into high-risk indicating that development proceeds from primary hemato-patients following high-dose therapy. We report that human poietic sites to the periphery. Repopulation of secondaryumbilical cord blood cells, 78% to 100% of which express recipients with human cells by BM from primary recipientsthe hematopoietic progenitor cell surface marker CD34, can demonstrates the maintenance of substantial proliferationconsistently engraft, develop, and proliferate in the hemato- capacity of the input precursor population. These data sug-poietic tissues of sublethally irradiated NOD/LtSz-scid/scid gest that the cells capable of initiating human cell en-mice. Engraftment and development of CD34" cells is not graftment (SCID-repopulating cells) are contained in thedependent on human growth factor support. CD34" cells CD34" cell fraction, and that this mouse model will be usefulhome to the mouse bone marrow (BM) that becomes the for assaying the developmental potential of other rare hu-primary site of human hematopoietic development con- man hematopoietic cell fractions in vivo.taining myeloid, lymphoid, erythroid, and CD34" progenitor

� 1997 by The American Society of Hematology.populations. Myeloid, and in particular lymphoid cells pos-

such as umbilical cord blood (CB) and mobilized peripheralM blood (PB) progenitor cells are currently under investigation.AMMALIAN HEMATOPOIESIS is a developmentalprogression that proceeds from a small pool of

Additionally, the possibility of expanding primitive stemunique progenitor cells with long-term, multilineage poten-cells ex vivo is potentially a clinically useful strategy thattial to larger populations with more limited potential, to theis actively being pursued in many laboratories.8,9 A majormature blood cells found in the circulation.1 Characterizationconcern with a number of these approaches is that the qualityand quantification of these progenitor pools is fundamentaland number of stem cells in these populations are largelyto our understanding of this developmental sequence and isunknown. Consequently an experimental transplantationof great importance for human hematopoietic cell trans-assay that could measure the repopulating potential of vari-plantation biology. Much of our understanding of the devel-ous human precursor fractions would be invaluable in fur-opment and regulation of the mammalian hematopoietic sys-thering our understanding of these primitive hematopoietictem, including stem and progenitor cell potential and lineageprogenitors.relationships, has been derived from work in the mouse.1 InOver the past decade, several groups have transplantedparticular, the transplantation assays available in the mouse

hematopoietic precursors into different mouse mutants in anmodel have been instrumental in defining and characterizingattempt to develop a reproducible transplantation assay. Inthe most primitive elements of the hematopoietic system;the SCID/hu model developed by McCune et al,10 a humannamely the long-term repopulating stem cell endowed withhematopoietic microenvironment is created by implantingthe potential to provide long-term, multilineage hematopoie-fragments of human fetal thymus and liver10 or bone11 intosis following transplantation into appropriate recipients.2-4severe combined immunodeficiency disorder (C.B-17-scid/As comparable experimental transplantation assays do notscid; SCID) mice. Hematopoietic precursor populations sub-exist for human hematopoietic precursors, little is known

about human long-term repopulating stem cells. The largemajority of studies reported to date have attempted to extrap- From the Bone Marrow Transplant Program, the Division ofolate in vivo performance of a population from results of in Medical Oncology, and the Department of Immunology, Universityvitro studies. Assays such as the colony forming unit-blast of Colorado Health Sciences Center, Denver; National Jewish Cen-

ter for Immunology and Respiratory Medicine, Denver, CO; Amgen,(CFU-blast),5 the high proliferative potential colony formingThousand Oaks, CA; the Department of Molecular and Medicalcell (HPP-CFC),6 and the long-term culture-initiating cellsGenetics, University of Toronto and the Department of Genetics,(LTC-IC)7 demonstrate the ability of a cell fraction to formResearch Institute, Hospital for Sick Children, Toronto, Ontario,colonies of erythroid, granulocyte/monocyte, and macro-Canada; and The Jackson Laboratory, Bar Harbor, ME.phage precursors over an extended time period in a defined, Submitted December 2, 1996; accepted February 17, 1997.

in vitro environment. However, a correlation has not been Supported in part by Grants No. P-20 CA66207 (to C.J.H.), RO1reported between these in vitro assay results and the reconsti- CA6508 (to E.J.S.), A130389 (to L.D.S.), and a Cancer Center Coretution potential of the same cell fraction in vivo in a human grant (CA 46934) from the National Institutes of Health and/or

National Cancer Institute, Bethesda, MD.hematopoietic environment. A further concern with these inAddress reprint requests to Christopher J. Hogan, PhD, Divisionvitro assays is that they are unable to predict the homing

of Medical Oncology, University of Colorado Health Sciences Cen-potential of particular populations.ter, Box B-171, 4200 E Ninth Ave, Denver, CO 80262.Gaining a better understanding of the human long-termThe publication costs of this article were defrayed in part by pagerepopulating cell is essential as hematopoietic cell-sup- charge payment. This article must therefore be hereby marked

ported, high-dose therapy is being employed with increasing ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely tofrequency for a broad spectrum of high-risk malignancies indicate this fact.and hematologic diseases. As a means of improving this � 1997 by The American Society of Hematology.

0006-4971/97/9001-0039$3.00/0therapy, potential new sources of hematopoietic stem cells

85Blood, Vol 90, No 1 (July 1), 1997: pp 85-96

AID Blood 0030 / 5h38$$$581 08-14-97 10:29:10 blda WBS: Blood




HOGAN ET AL86

National Jewish Center (NJC; Denver, CO) from breeding pairssequently injected into these transplanted fetal tissues havekindly provided by Leonard D. Shultz. The breeding colony isbeen shown to proliferate and differentiate.11-13 This has beenhoused in a restricted barrier facility and the experimental animalsa particularly useful model for examining lymphocyte devel-were maintained in microisolator cages on laminar flow racks in aopment in vivo.10,12 However few of the experimental cellsclean experimental room. Mice were maintained on an irradiated,in these models seed the mouse bone marrow (BM) or the sterile diet of Picolab Mouse Diet 20 (PMI Feeds, Inc, St Louis,peripheral hematopoietic tissues; instead they are generally MO) and given autoclaved, acidified water.

restricted to the fetal explants. Models where human hemato- NOD/SCID mouse manipulations. All animal experiments werepoietic cells are transplanted intravenously into sublethally approved by the Animal Care Committees of the NJC and the Uni-irradiated mice may more closely recapitulate the ability of versity of Colorado Health Sciences Center. Immediately before cell

transplantation 8- to 10-week-old NOD/SCID mice were injectedparticular human progenitors to home to primary hematopoi-intraperitoneally (IP) with 250 mL of phosphate-buffered salineetic sites as well as reflect their in vivo developmental poten-(PBS) that contained 50 mL of reconstituted anti-asialo GM1 (Wakotial. Such systems have been developed in beige/nude/x-Chemicals, Inc, Richmond, VA). Identical treatments were per-linked immunodeficiency (BNX)14-17 and SCID18-22 mice.formed on days 5 and 11 postinfusion of experimental cells. MiceUnfractionated adult BM,14,17,19,20 PB,17,20 and CB,21 as wellwere sublethally irradiated with 350 to 400 cGy from a 137Cs sourceas fetal BM,17,22 have engrafted and been maintained for immediately before intravenous tail-vein injection of 250 to 300 mLvarying lengths of time in these animals. Maintenance of of Iscove’s modified Dulbecco’s medium (IMDM; GIBCO, Grand

human cells in the mouse BM and periphery suggests human Island, NY) with 10% fetal bovine serum (FBS; Sigma Chemicalhematopoietic development can occur in the mice. Further- Co, St Louis, MO) that contained an appropriate number of CBmore, Nolta et al15,16 have shown that when BM- or PB- cells from the cell fraction being tested. In cases where secondaryderived CD34/ progenitor cells were cotransplanted with transplants were performed, BM cells were prepared from primary

recipients as described below, pelleted 4 minutes at 720g and resus-human interleukin-3 (IL-3)–expressing stromal cells intopended at 1.6 to 2.4 1 106/mL in IMDM/10% FBS. SecondaryBNX mice, they could be maintained and generate cells ofrecipients each received 4 to 6 1 106 cells of the mouse/human cellboth the myeloid and lymphoid lineages. In most of thesesuspension. For the duration of selected experiments, mice werestudies, high frequencies of engraftment or hematopoieticinjected IP three times per week with 250 mL PBS/5% FBS con-development have depended on treatment of mice with hu-taining 10 mg each of the human growth factors IL-3, granulocyte-man cytokines or cotransplant with stromal cells engineered macrophage colony-stimulating factor (GM-CSF), and stem cell fac-to produce human cytokines. In studies where growth factors tor (SCF) (Amgen, Inc, Thousand Oaks, CA). After infusion of cells,

were not used,17,21,22 high numbers of unfractionated input mice were maintained in a HEPA-filtered isolator (Isotec, Bichester,cells were required and/or engraftment was infrequent.17 UK) within an ultraclean barrier room for the duration of the experi-Recently, the scid mutation has been backcrossed onto the ment. The mice were killed in a CO2 chamber at predetermined time

nonobese diabetic (NOD/Lt) mouse background. In addition points. Blood was collected into heparinized tubes from poolsformed in the chest cavity after the dorsal aorta was severed. Femursto lacking T- and B-cell function, the resulting NOD/LtSz-and tibia were collected and aspirated with PBS/5% FBS to liberatescid/scid (NOD/SCID) mice exhibit low natural killer cellBM cells. Cell suspensions were then filtered through sterile 70 mmactivity, are defective in macrophage function, and lack he-Nitex to get rid of clumps and debris. The spleen and thymus (ifmolytic complement.23 NOD/SCID mice have shown im-present) from each mouse was harvested and strained through sterileproved engraftment over the SCID model when transplanted70 mm Nitex into PBS/5% FBS to collect cells.with unfractionated human spleen cells,24 BM leukocytes,25 CB CD34/ cell enrichment. Blood was aseptically aspiratedPB mononuclear cells,26 and unfractionated CB cells.27,28 from placenta and umbilical CB veins immediately following normal

The improved ability to transplant human hematopoietic cells obstetrical deliveries at University Hospital (Denver, CO) and placedinto NOD/SCID mice provides the foundation to develop a into blood bags containing an anticoagulant (citrate, phosphate, dex-repopulation assay for primitive human cells. Although previ- trose, adenine-1). Samples were processed within 8 hours of collec-ous work has suggested that the cell capable of initiating the tion. Mononuclear cell (MNC) fractions were prepared by diluting

blood 1:1.5 in PBS containing 300 U/mL DNase I (Sigma) and 1engraftment of human cells (termed the SCID-repopulating cell;mmol/L MgCl2, layering onto Ficoll (Lymphocyte Separation Me-SRC) is primitive,29 little work has been done to characterizedium, Organon-Technika, Durham, NC) and centrifuging for 30the SRC. As such, this report is an initial characterization ofminutes at 500g. The cells were washed in a buffer (PBS, 1 mmol/hematopoietic development in NOD/SCID mice from the hu-L MgCl2, 100 U/mL DNase I, and 1% human serum albumin),man CD34/ progenitor cell fraction of CB. Evidence is pre-resuspended in the buffer and counted. CD34/ cell fractions weresented that CD34/ CB cells can consistently home to the BM prepared from ficoll-separated MNCs using the buffer describedand engraft in NOD/SCID mice. Furthermore, engrafted above and the CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec,

CD34/ populations can develop and give rise to myeloid and Inc, Sunnyvale, CA) as per the manufacturer’s directions. Cells werelymphoid cell lineages without the influence of exogenously fractionated on a MACS column Type RS using a VarioMACS cellsupplied human growth factors. In addition, cells from the separator (Miltenyi Biotec, Inc). The eluted CD34 fraction wasCD34 fraction with short-term myeloid progenitor potential are passed over a second RS column to obtain greater enrichment ofdetected throughout the course of engraftment, while SRCs CD34/ cells.

Flow cytometry. At least 5 1 104 nucleated cells were treatedcapable of repopulating secondary recipients are maintained forfor lysis of erythrocytes with 1 mL hemolytic buffer (155 mmol/Lextended periods of time.NH4Cl, 12 mmol/L NaHCO3, 0.1 mmol/L EDTA) at room tempera-

MATERIALS AND METHODS ture for 2 minutes followed by washing with PBS/0.1% bovine serumalbumin (BSA) and centrifugation at 500g for 5 minutes. To preventMice. Experimental NOD/SCID mice were obtained from a

breeding colony established at the Biological Resource Center at the nonspecific antibody binding, cells were resuspended for 20 minutes





CORD BLOOD CD34/ CELL DEVELOPMENT IN MICE 87

at 4�C in an antibody against Fc receptor (24.G230). Cells were 72�C, 45 seconds, and a final primer extension at 72�C for 7 minutes.Specific primers included: 5�-aaggataccacaataagctgc-3�, and 5�-washed and pelleted before being resuspended in 5 mg/1 1 106 cells

of the appropriate antibody and incubated for 20 minutes at 4�C. ggtttgtggagactggcac-3�. This primer set yields a 156-bp product fromthe 3� untranslated region of the human Cart-1 gene and fails toCells were then washed in PBS/0.1% BSA and resuspended in 0.5

mL of the same buffer. Two- or three-color flow cytometry was amplify a product from mouse genomic DNA.33 Positive and nega-tive controls for human specificity of the primers included usingperformed with a Coulter EPICS XL flow cytometer (Coulter, Hia-

leah, FL) using the software provided. Cells from a control mouse template extracted from either 1,000 human PB white cells or 1,000mouse BM cells, respectively. As a control for the presence ofwere stained with the same antibodies as a negative control. Appro-

priate isotype controls were also run for each fraction analyzed. Anti- sufficient DNA template, parallel PCR reactions were performedon each sample using primers specific for b-actin. This primer sethuman (hu) CD45-fluorescein isothiocyanate (FITC) and antimouse

(mu) CD45-phycoerythrin (PE) were used to identify the ratio of consisted of: 5�-aaggccaaccgcgagaagat-3� and 5�-tcggtgaggatcttcat-gag-3�, that amplifies a 249-bp fragment from control genomichuman:mouse leukocytes. Specific subsets of human cells were

quantified by gating on hu CD45-PerCP/ cells and then assessing DNA.34 The reaction conditions were identical to those describedabove except TaqStart antibody was not used.staining with anti-hu: CD34-PE (multilineage progenitors), CD33-

PE (myeloid progenitors) and CD13-FITC (myeloid), CD14-PE(monocytes) and CD42a-FITC (megakaryocytes), CD19-PE (B cells) RESULTSand CD2-FITC (T cells), CD19-PE and IgM-FITC (developing B

Phenotype of input cells. To obtain highly enrichedcells). Developing T cells were quantified by gating on hu CD3-CD34/ cell fractions for use as input cells, double immuno-PerCP/ cells and then assessing staining of anti-hu: CD4-FITC and

CD8-PE. Nucleated human erythroid progenitors were assessed by selection was performed on most fractions. In two experi-quantifying hu CD71-FITC//CD45-PerCP0 cells in the hu glycopho- ments (5 and 8), the cells were immunomagnetically frac-rin A-PE/ gate. A four parameter flow methodology was used for tionated once and then purified further by fluorescence-quantification of all CD34/ populations.31 All antibodies were from activated cell sorting (FACS). Typically the yield followingBecton Dickinson (San Jose, CA), except for anti-CD13 and anti- immunomagnetic selection from a single CB was in theglycophorin A (Immunotech, Westbrook, ME), anti-mu CD45 (Phar- range of 0.5 to 2.0 1 106 cells and thus limited the amountmingen, San Diego, CA) and anti-hu IgM (Caltag, So. San Francisco, of input cell phenotyping that was feasible. To determineCA).

the phenotype of input cells, four CB samples each wereCell sorting. Staining of CD34-selected fractions was performedfractionated by double immunomagnetic selection and theas described above using anti-hu: CD34-PE, CD19-FITC, and eitherresulting populations (Control CB fractions in Table 1) wereCD2-FITC or CD3-FITC. Cells were sorted aseptically into CD34/

CD190/20(30) and CD34/ CD19//2/(3/) fractions with a Coulter used to assess the frequency of cells bearing the surfaceEPICS 752 flow cytometer using a Cicero acquisition system and markers subsequently measured in output cells from the ex-Cyclops software (Cytomation, Ft Collins, CO). periments. Antibody combinations and flow gating used forCFU assay. Input cells, as well as BM cells from experimental analysis were identical to those used for experimental output

mice at the time of death, were placed into a CFU assay. The number cells. CD34/ cell purity ranged from 75% to 95% in controlof mouse BM cells plated ranged from 1.47 to 3.57 1 105/plate, fractions (Table 1). Purities of CD34/ fractions used in trans-depending on the human cell content. One-milliliter aliquots of a plant experiments fell within this range or were above itmixture containing (vol/vol): 44% methylcellulose (Terry Fox Labo-

(Table 1). Table 1 shows that myeloid progenitor cellsratories, Vancouver, BC), 30% FBS, 10% BSA, 1% 0.1 mmol/L(CD33/) comprised a consistent, albeit low, percentage ofmethylprednisolone, 1% 11 mmol/L b-mercaptoethanol, and 14%CD34 fractions, while more mature myeloid cells (CD13/IMDM containing the appropriate number of cells, plus 1 U/mL

human erythropoietin, 10 ng/mL each of human: IL-3, GM-CSF, and CD14/) were largely absent. Development from CD34/and SCF (Amgen, Inc) were plated in quadruplicate into 30 cm2 cells into the myeloid lineages could thus be detected in micedishes (Falcon 1008; Becton Dickinson, Lincoln Park, NJ) as pre- using these fractions. Erythroid progenitor cells (glycophorinviously described.32 After 14 to 15 days at 37�C, the plates were A/CD71/CD450) were present as well and ranged fromscored for colony forming cells that included granulocyte/monocyte 0.15% to 3.8% of the fractions. The major contaminants in(CFU-GM), burst-forming units-erythroid (BFU-E), and granulo- CD34 fractions were B cells (CD19/) and T cells (CD2/). Tocyte/erythroid/megakaryocyte/macrophage (CFU-GEMM). distinguish between clonal expansion of CD19/ or CD2/(3/)Polymerase chain reaction (PCR) for hu Cart-1. Individual col-

lymphoid cells and lymphoid development from CD34/ cellsonies for analysis were plucked from CFU plates and placed intoin mice, CD19/ and CD2/(3/) cells were eliminated fromtubes containing PBS. Cells were washed once in PBS and resus-two input cell fractions (experiments 5 and 8; see Table 1)pended in 25 mL 0.11 PBS in PCR tubes. The tubes were heated

to 95�C for 8 minutes in a thermocycler. One mL proteinase K (20 by FACS.mg/mL; Boehringer Mannheim, Indianapolis, IN) was added to each Engraftment into mice. All CD34/ cell-enriched frac-tube and the tubes were placed back in the thermocycler and held tions used to transplant NOD/SCID recipients to date haveat 56�C, 30 minutes and then at 94�C, 8 minutes. Tubes were spun successfully engrafted the mice (Fig 1). Short-term hemato-and supernatant used for PCR reactions. PCR reactions contained poietic development was analyzed after 6 to 10 weeks in11 PCR buffer (Perkin-Elmer, Branchburg, NJ), 1.5 mmol/L MgCl2, most experiments, although engraftment persisting through0.1 mmol/L nucleotide triphosphates, 0.125 mL AmpliTaq polymer- 15 weeks was demonstrated in experiment 7 (Fig 1). Inase (Perkin-Elmer) preincubated for 5 minutes at room temperature

several experiments the extended engraftment potential ofwith 0.138 mg TaqStart antibody (Clontech, Palo Alto, CA), 1 mmol/CD34 fractions was assessed by serial transplantation of BML of each specific primer, 10 mL sample, and dH2O to make a finalfrom primary recipients (see below). Although the BM wasreaction volume of 25 mL. The ‘‘hot start’’ reaction was performedthe primary site of human cell engraftment and proliferation,in a Perkin-Elmer 9600 and included a pre-melt for 2 minutes at

94�C followed by 30 cycles of 94�C, 30 seconds, 58�C, 1 minute, human cells were also detected and analyzed in the spleen





HOGAN ET AL88

Table 1. Phenotype of CD34-Enriched Cell Fractions

Percent Cells in Fraction

Control CB* Experiment†

Cell Surface 1 2 3 4 1 2 3 4 5‡ 6 7 8‡

CD34/ 95 92 89 75 85 92 95 78 100 85 99 100CD33/ 1 1.1 0.2 2.0 ND ND ND ND ND ND ND NDCD13/ 0 0 0 0 ND ND ND ND ND ND ND NDCD14/ 0 1 0.6 1.2 ND ND ND ND ND ND ND NDCD19/ 1.2 2.3 1.8 6.2 ND ND ND 1.9 0 ND 6.4 0CD2/ ND 2 5.2 13.9 ND ND ND 1.2 0 ND 0.6 NDCD3/ ND ND ND ND ND ND ND ND ND ND ND 0glyc A/ CD71/ CD450 0.6 0.15 2.9 3.8 ND ND ND ND ND ND ND ND

Abbreviation: ND, not determined.* Cell populations in CB fractionated by double immunomagnetic selection.† Cell populations in fractionated CB used to engraft mice.‡ Cells were fractionated by one round of immunomagnetic selection followed by FACS for the CD34/ CD190/20(30) fraction.

and PB of all recipients. The fraction of human cells in these 2.75 1 104 CD34/ cells (experiment 8) were capable oforgans ranged from 1% to 30% and was always lower than yielding detectable engraftment levels.that detected in BM. In two mice (from experiments 1 and Conditions were assessed that affected engraftment of3), the thymus was found to contain human cells exclusively. CD34 fractions into recipients such as the pretreatment radia-It should be stressed that repopulation of mice thymi with tion dose and the need for administration of human growthhuman cells was not a consistent finding, but rather, a rare factors. Initial experiments were performed by irradiatingevent. Although a limiting dilution experiment was not per- mice with 400 cGy. At this dose 65% (26 of 40; experimentsformed, the level of engraftment generally fell off with de- 1, 3, and 7) of the animals died 1 to 2 weeks posttransplant.creasing numbers of input cells (Fig 1). However, as few as Surviving mice generally engrafted well, however acute

deaths, most likely due to radiation-induced toxicity, wereconsidered too extreme. In experiments where mice received350 (experiments 2 and 4 through 6) or 375 (experiment 8)cGy, engraftment levels were slightly lower than in micethat had received higher doses, but the number of earlydeaths was dramatically reduced to 20% (eight of 40). Forlater experiments, 350 cGy was used routinely for pretreat-ment.In previous studies, consistent engraftment and develop-

ment of CD34/ cells has relied on the influence of humangrowth factors.15,16 Alternatively, unfractionated CB (com-prised of cells known to produce growth factors) has beenshown to engraft mice to comparable levels with and withoutgrowth factor supplements.21 Two experiments were per-formed (1 and 2) to assess the influence of human: IL-3,GM-CSF, and SCF supplied three times per week on en-graftment and subsequent development of CD34 fractions.Figure 1 illustrates that engraftment levels in mice that re-ceived no growth factor treatments (open circles) were com-parable to those in mice that received growth factors (closedcircles). Additionally, significant differences were not de-tected in output human cell subpopulations between treatedand untreated animals (Fig 2 and see below). Because ofthese findings, subsequent experiments were done withoutthe use of growth factors.Fig 1. Engraftment of human CD34" cell-enriched fractions intoHematopoietic development. BM, spleen, and PB wereNOD/SCID BM. The percent of huCD45" cells found in NOD/SCID BM

are plotted for individuals in experiments 1 through 8. The number harvested from mice 6 to 15 weeks after infusion of CBof input cells, pretreatment radiation dose, and duration of the exper- CD34/ cells and the fraction of human cells present in eachiment are listed below each experiment number. In experiments 1 hematopoietic tissue was determined by costaining cells withand 2, 10 mg each of human: IL-3, GM-CSF, and SCF was administered

antibodies specific for both mouse and human CD45, respec-three times per week to one group of mice (●), while others weregiven no growth factors (�). tively. The cellularity of the tissues in all recipients was






Fig 2. Summary of huCD45"

cell subpopulations found inmouse BM (A), spleen (B), andPB (C). The percent of thehuCD45" population that alsoexpressed human myeloid cell(CD13/33" and CD14"), B cell(CD19"), erythroid progenitorcell (gA"71"45Ï), and CD34" cellsurface markers is shown for allsamples analyzed from experi-ments 1 through 8. Median val-ues are shown above eachsubpopulation. Samples are iden-tified from experiments 1 (�, �)and 2 (�, �) where mice receivedeither 10 mg each of human: IL-3, SCF, and GM-CSF, three timesper week (�, �), or received nogrowth factor treatments (�, �).

normal. At the levels of engraftment obtained (Fig 1), a from input CD34/ populations. Cells within the huCD45gate were quantified for myeloid, lymphoid, and erythroidsignificant expansion of human cells in the mice occurred

over the course of the experiments. For instance, the BM cell surface markers, as well as for the retention/expansionof a CD34/ population. Figure 3A shows representative anal-(from two femurs and two tibia) of an individual mouse in

experiment 4 contained 3.25 1 107 cells, of which 30% were ysis of BM, spleen, and PB, while Fig 2 summarizes thevalues of each huCD45/ subfraction for all samples analyzedhuman. Thus, 9.75 1 106 human cells in the femurs and

tibia were derived from 1 1 105 input cells, representing an in experiments 1 through 8. The myeloid component of BMranged from 5% to 57% of the human population. CD13/increase of at least 500-fold (the femurs and tibia represent

only 20% of the BM cavity35). In general, increases of human and CD13//33/ cells made up the majority of these popula-tions, while CD14/ cells comprised a smaller proportion ofcell numbers were observed in all mice, indicating that the

mouse environment was conducive to proliferation of human huCD45/ cells. The fraction of myeloid cells in thehuCD45/ gate varied between experiments, as well as be-hematopoietic cells.

Cells from the tissues were further analyzed by three-color tween individuals within experiments. B cells were the majorhuman component of BM, in most cases, comprising 8.4%flow cytometry for evidence of multilineage development





HOGAN ET AL90

Fig 3. Development of hu-man CD34" cells in the hemato-poietic tissues of NOD/SCIDmice. (A) Typical profiles of hu-man progenitor cell (CD34,CD33), myeloid cell (CD13,CD14), B-cell (CD19), and T-cell(CD2) surface markers onhuCD45" output cells from theBM, spleen, and PB of repopu-lated mice. Cells in the glycopho-rin A" gate were 100% huCD71"

and 0% to 3% huCD45". Quanti-fication of the CD34" populationwas done by using a four param-eter gating method.31 (B) In therare cases where huCD45" cellspopulated the thymus (twomice), the vast majority (98%)were huCD2" (upper histogram).When the thymic populationwas gated on huCD3" cells(which comprised 72% of the to-tal cells), 95% were hu CD4/CD8double positive cells (lower his-togram).

AID Blood 0030 / 5h38$$0030 08-14-97 10:29:10 blda WBS: Blood





to 85% of the huCD45/ cell population. Erythroid progeni- CD14/ cells ranged from 2.6% to 8.4%, and CD34/ cellscomprised 2% to 18.6% of the huCD45/ population. Impor-tors were consistently found in low frequencies. BM from

the majority of mice analyzed contained a substantial propor- tantly, CD19/ cells were the major population of humancells in the BM, spleen, and PB of all animals (Fig 4). Intion of CD34/ cells. The proportion of CD34/ cells did not

depend on the duration of the graft. Mice analyzed at 6 BM, CD19/ cells accounted for 28% to 62% of the humanleukocytes. These cells were derived in the mice from SRCsweeks had 1.5% to 6.3% CD34/ cells, while mice analyzed

at 15 weeks retained 2.5% to 6.9% CD34/ cells in their within a CD34 fraction containing cells with no detectableexpression of CD19, suggesting that cells in this system canBMs.

Figures 2 and 3A reveal that CD13/ and CD14/ myeloid differentiate down the lymphoid pathway from CD34/ cellsdevoid of an early B-cell surface marker.cells were consistently found in the spleen and in the periph-

eral circulation of recipients, albeit generally at lower levels The huCD19/ populations of BM, spleen, and PB wereanalyzed for cell surface expression of hu IgM as evidence ofthan that seen in BM. Much lower proportions of myeloid

cells were found in the spleen than in PB. As in BM, a further B-cell development. For this analysis, human CD45/BM, spleen, and PB cells from repopulated mice weregreater proportion of huCD45/ cells expressed CD13 than

CD14, while the majority of cells expressed CD19 (Figs 2 stained with both anti-hu CD19 and anti-hu IgM. HumanCD45/ cells from the lymph gate of the light scatter profileand 3A). It should be noted that the myeloid compartment

seen in these organs consisted of CD13/ and CD14/ cells are shown in Fig 5. Without exception, all mice analyzedshowed similar patterns of expression in the three hemato-and generally lacked the CD33/ and CD13//33/ populations

that were found in BM (compare histograms in Fig 3A). poietic compartments. This included mice that had beentransplanted with CD34/CD190 cells from experiments 5Erythroid progenitors were found in over half of the spleens

analyzed and were generally low in frequency. Erythroid and 8 where both more mature CD19/ cells, as well asCD34/CD19/ pro B cells were absent from the input cellprogenitors were not detected in PB. CD34/ progenitor cells

were consistently found in spleen at lower frequencies than fraction (Table 1). In the BM, the majority of CD19/ cellsexpressed no cell surface IgM; a finding that would be ex-in BM. Again, no CD34/ cells were detected in PB samples

(Figs 2 and 3A). Figure 2 also shows that there were no pected in a primary lymphopoietic site. In contrast to theBM, approximately two thirds of the CD19/ cells in thesignificant differences in subpopulation distributions found

in BM, spleen, or PB samples between mice supplemented spleen expressed IgM, indicating a more mature B-cell popu-lation. In PB, 75% to 90% of the CD19/ population ex-with human growth factors and those that were not (experi-

ments 1 and 2). pressed IgM. These findings suggest that human B-cell matu-ration in mice is correlated with a progression of cells fromA grossly visible thymus was usually absent from mice

at the time of killing. In mice where the thymus was seen, the site of primary lymphopoiesis, the BM, to the peripheralcirculation, similar to the situation in humans.it was collected and analyzed. Most contained mouse CD45/

cells exclusively that were probably a prelude to thymoma, Colony-forming potential in vitro. As a further indicationof human myelopoiesis, the human hematopoietic colony-form-which develops in these mice after 5 to 7 months of life.23

However, two mice (one each from experiments 1 and 3) ing potential was determined for output cells from the BMs ofrepopulated mice. The CFU assay was performed on outputhad a thymus that contained 1 to 2 1 107 cells that were

91% to 96% huCD45/. Within this population, 98% of the cells from mice in all experiments. BM from all mice exhibitedthe same levels of output cell colony-forming potential relativecells were CD2/ (Fig 3B, top histogram). When cells in this

population were stained with anti-hu: CD3, CD4, and CD8, to that of input cells, including those mice that were treated withhuman growth factors. Data from experiment 4 are presented init was found that the majority (95%) were CD3//CD4//

CD8/ (Fig 3B, bottom histogram). Input cells consistently Fig 6 as representative. A substantial number of CFUs, thatincluded CFU-GM, BFU-E, and CFU-GEMM, were found inwere contaminated with a CD2/ population (Table 1). How-

ever, analysis of two CD34/ input cell fractions showed the the BMs of all mice analyzed (Fig 6, open circles). Values fortotal CFUs were calculated by multiplying the number of CFUspresence of no CD4/CD8 (double positive) cells, suggesting

the human T cells found in these thymi represent de novo counted by the fraction of total huCD45/ cells/huCD45/ cellsplated for both input cells and output cells from each mouse.lymphopoiesis in the mouse thymic environment. The basis

for the inconsistent nature of thymic colonization by human Figure 6 (closed circles) reveals that the total colonies derivedfrom 1 1 105 input cells included 2.09 1 103 CFU-GM, 5.88CD34/ cells remains to be determined.

As lymphoid cells were a consistent contaminant of input 1 103 BFU-E, and 30 CFU-GEMM. Total output cell CFUswere calculated for BMs of five mice (range of huCD45/ cellcell CD34 fractions, the CD19/ cells observed in mouse

hematopoietic tissues might have been due to expansion of populations, 14% to 34%) 10 weeks postinfusion of 1 1 105input cells and included (mean { standard error [SE]): 1.6 1cells already committed to the B-cell lineage. To determine

if CD19/ cells were indeed generated from more immature 104 { 3.4 1 103 CFU-GM, 2.08 1 104 { 3.05 1 103 BFU-E,and 176 { 29 CFU-GEMM (Fig 6, open circles). These areprecursors, CD19/ cells were removed from CD34 fractions

by FACS before infusion into mice (experiments 5 and 8). the minimum number of human CFUs derived from the mice;cells capable of producing myeloid colonies that were not takenAnalysis of these experiments showed that myeloid,

lymphoid, erythroid, and progenitor cell levels in BM, into account included the BM fraction excluded from the fe-murs and tibia and the spleen cell population.spleen, and PB fell within the range of values seen in the

other experiments. CD13//33/ cells made up 6% to 26%. To confirm that the colonies grown in the CFU assay were





HOGAN ET AL92

Fig 4. CD19" B-cell develop-ment from CD34"CD19Ï inputcell populations in NOD/SCID re-cipients. Elimination of contami-nating CD19" cells by FACS frominput CD34 cell fractions had noeffect on the generation of B-cellprecursors in the mice. The his-tograms show that, as in otherexperiments, output cells ex-pressing CD19 predominated inthe BM, spleen, and PB of repop-ulated mice.

of human origin, all GEMM colonies, as well as 30 GM and modal proportions and using 100% as the estimated propor-tion and analysis of at least 60 colonies/mouse, the confi-30 BFU-E colonies (representing 3.5% to 10.6% and 5.4%

to 8.4% of the total GM and BFU-E colonies, respectively, dence level is 95% that from 94% to 100% of the coloniesfrom each mouse were human. These results strongly suggestderived from each mouse) were plucked from CFU plates

of each mouse and analyzed by a human-specific PCR (Fig that only human colonies were able to proliferate under theconditions used in this CFU assay.6). While DNA extracted from untransplanted, control

mouse BM cells showed no amplified product, every colony Taken together, these data suggest that substantial humanmyelopoiesis and erythropoiesis occurs in mouse BM asanalyzed from output cell CFU plates produced a product

of the same size as one amplified from human white blood demonstrated by the significant number of human colony-forming cells identified.cells (É150 bp; Fig 6). Using an exact nomagram for bi-

Fig 5. B-cell development. In the BM of mice fromall experiments only 10% to 20% of huCD19" cellsexpressed cell surface IgM. In the spleen 60% to 75%of CD19" cells also expressed IgM. PB from all micerevealed that 75% to 90% of huCD19" cells also ex-pressed IgM. This was true for mice transplantedwith input CD34" cell fractions known to be contami-nated with low levels of CD19" cells (experiments 4and 7, Table 1), as well as for mice transplanted withCD34" input fractions devoid of any CD19" cells (ex-periments 5 and 8, Table 1).






Fig 6. Human myeloid colony-producing poten-tial in vitro of input cell fractions and of output cellsfrom the BMs of repopulated mice. Total GM, ery-throid (BFU-E), and GEMM colonies are plotted for 1Ì 105 input cells from experiment 4 (●). BM cellsfrom five mice that had each been infused with 1 Ì105 input cells and allowed to engraft for 10 weekswere plated into the CFU assay. The means and SEof the total CFU in each category are plotted (�). Allcolonies analyzed by a human-specific PCR (at least30 colonies/CFU type/mouse) were of human origin.Typical PCR signals are shown below specific colonytypes for human Cart-1 (lanes 6, 8, and 10) and forb-actin (lanes 7, 9, and 11). Controls consisted ofPCRs for human Cart-1 (lanes 2 and 4) and b-actin(lanes 3 and 5) from human PB leukocytes (lanes 2and 3) and normal mouse BM cells (lanes 4 and 5).Lane 1 shows a 123-bp ladder. The predicted ampli-fied products for the hu Cart-1 PCR and for the b-actin PCR are 156 and 249 bp, respectively.

Secondary transplants. As phenotypic analysis of pri- expressed CD14, and no CD34/ cells were detected (notshown). Human CD45/ cells in the PB consisted of CD13/mary recipients indicated maintenance of a human CD34/

population, the next series of experiments was aimed at de- (13%), CD14/ (5.7%), and CD19/ (61%) populations (notshown). SRCs in the original CD34-enriched fraction, there-termining whether the human cell fraction contained SRC

with the capacity for more durable, multilineage en- fore, were capable of sequentially repopulating two micewith myeloid, lymphoid, and progenitor cells over a totalgraftment. In these experiments, we evaluated the ability of

BM from previously engrafted mice to repopulate secondary period of 4 months.Both mice transplanted with mouse BM from experimentrecipients with human cells. BM of one mouse from experi-

ment 1 (83% huCD45/ after 8 weeks) was transplanted into 7 survived and were analyzed 9 weeks posttransplant. BMsfrom the mice contained 10% and 6% huCD45/ cells, re-three recipients (calculated human CD34/ cells received/

mouse was 6.8 1 105) and BM of a second mouse, from spectively, while spleens and PBs from both mice contained1% to 2% huCD45/ cells. Subpopulation analysis ofexperiment 7 (51% huCD45/ after 15 weeks), was trans-

planted into two recipients (calculated human CD34/ cells huCD45/ cells showed results similar to those describedabove. To date, the original CD34-enriched fraction has beenreceived/mouse was 2.1 1 105). Two of the three mice from

the first transplant died 7 and a half weeks posttransplant, capable of engrafting and developing in mice for 4 monthswhile maintaining cells able to repopulate secondary recipi-however significant human engraftment was found in the

BM and periphery of the remaining mouse 8 weeks post- ents for an additional 2 months, resulting in a total en-graftment time of 6 months. Although only a limited numbertransplant. Human CD45/ cells comprised 23% of BM cells,

5% of spleen cells, and 5.5% of PB cells in this mouse (Fig of secondary recipients have been analyzed, these data sug-gest that cells maintained in primary recipients did retain7A). Within the huCD45/ gate of the BM the levels of

developing progenitor, myeloid and lymphoid cells paral- some repopulation capacity.leled those seen in primary transplants. Specifically, 23%

DISCUSSIONexpressed CD13/33, 8% expressed CD14, 63% expressedCD19, and 5.5% expressed CD34 (Fig 7B). Thus, a CD34/ Evidence presented in this report establishes that CD34-

enriched cells derived from human CB and infused intrave-population was maintained at levels equal to those in primaryrecipients. In the spleen, the majority of huCD45/ cells ex- nously consistently engraft NOD/SCID recipients. Over 90%

of recipients that survived to the end of the experimentalpressed CD19 (80%), while 8% expressed CD13/33, 4%





HOGAN ET AL94

Fig 7. Human engraftment of secondary recipients by BM from a primary recipient of human CD34" cells. (A) Histograms of BM, spleen,and PB cells stained with antibodies against both mu and hu CD45. All secondary recipients from experiments 1 and 7 contained huCD45"

cells in these tissues. (B) Typical profiles of the myeloid, lymphoid, and progenitor cells comprising the huCD45" cell fraction of mouse BMare illustrated with histograms from the BM of a secondary recipient. Mouse BM cells were triple-stained and cells within the huCD45" gatewere analyzed. Populations of myeloid progenitors (CD33", 2.3%), early myeloid cells (CD13"/33", 11.3%), more mature myeloid cells (CD13",9.5%), monocytes (CD14", 8%), and B cells (CD19", 63%) were observed, while a significant proportion (5.5%) of huCD45" BM cells expressedCD34.

period showed varying degrees of human cell repopulation. of CB progenitors, as well as the output cell subpopulationdistributions and colony-forming potential, were largely un-As in human hematopoietic progenitor cell transplants, BM

was the primary site of repopulation, but peripheral hemato- affected by the influence of human IL-3, GM-CSF, and SCF.Previous studies have shown that unfractionated CB canpoietic organs showed significant repopulation with human

cells as well. This is the first report showing that the CD34/ engraft without the addition of exogenous growth factors.21However, this population contains cell types (eg, T cells)progenitor cell fraction of human CB can engraft and prolif-

erate throughout the hematopoietic tissues of NOD/SCID that could be producing growth factors in vivo. This is thefirst report in a mouse xenotransplant system where humanrecipients.

For mice to survive in reasonable numbers following suc- hematopoietic development has been seen from CD34/ pro-genitor cells without growth factor supplementation. This iscessful engraftment, the pretreatment radiation dose was cru-

cial. At 400 cGy, 65% of recipients died within the first 2 significant because high levels of human growth factors arenot normally found systemically and could alter the en-weeks following radiation treatment. This is perhaps not

surprising, as the SCID mutation is known to confer high graftment potential of the input cell population. This dataalso suggests that cells contained in the CD34/ CB popula-radiation sensitivity to the animals.36,37 However, at a dose

of 350 cGy, only É15% of the mice died acutely following tion possess an intrinsic ability for engraftment. Alterna-tively, or perhaps in addition, the NOD/SCID stroma mayirradiation. While at the higher radiation dose, surviving

mice generally showed the highest levels of engraftment (5% be a particularly favorable environment for supporting en-graftment of these cells.38 The fact that growth factor supple-to 95%), levels found at the lower radiation dose, where

huCD45/ cells in the BM generally ranged from 10% to ments are not required for engraftment, development, andproliferation of CD34/ cells may be advantageous for future60%, were adequate for the developmental analysis of human

cells. studies in determining the in vivo developmental potentialof rare human progenitor cell populations without the biasIn other mutant mouse models, human growth factor sup-

plements were required to obtain substantial proliferation of that specific growth factors may introduce.The human CD34/ cells infused into mice did nottransplanted human BM or PB cells.14-16,18-20 In this system,

engraftment and development of CD34/ CB cells in the randomly distribute and proliferate throughout the mousehematopoietic organs, but instead were partitioned in a man-absence of exogenous human growth factors was evident

over the entire range of input cell doses used. In two of the ner consistent with normal hematopoiesis. Human CD34/cells were found primarily in the mouse BM, as were theexperiments (1 and 2), where input CD34/ cell doses were

5 1 105 and 3.5 1 105, respectively, the level of engraftment most immature populations of B cells (predominantly






CD19/IgM0) and myeloid cells (CD13//33/). In addition, Likewise, repopulating SRCs with the potential to hometo and repopulate a secondary recipient are maintained inerythroid precursors were found primarily in the BM. In

mouse PB, more mature populations of myeloid (CD13/) NOD/SCID mouse BM for at least 4 months. Engraftmentlevels in secondary recipients were not as high (range, 6% toand B cells were found (predominantly CD19/IgM/), while

no populations of CD34/ cells or of erythroid progenitors 23% in BMs) as those generally found in primary recipients,however, this may be explained by the fact that human cellswere detected. This finding suggests not only that mouse

BM was the primary site of human hematopoiesis, but that in the input fraction had to compete with normal mouse BMcells for available sites in the recipients’ BM. Engraftedcells capable of engrafting mice in the original input CD34

fraction homed to the mouse marrow. Human CD34/ cells, human cells were once again capable of producing myeloidand lymphoid populations, as well as sustaining a CD34/at least to some extent, must recognize and adhere to the

appropriate environment in the mouse BM, suggesting that population for the duration of the experiments (an additional2 months). In an elegant experiment, Nolta et al16 geneticallythis homing mechanism may be conserved between these

species. Moreover, it appears that the developmental pro- marked input human CD34/ cells derived from BM and PBand showed that BNX mice retained multilineage repopulat-gression of human progenitor cells to more mature, lineage-

defined cells proceeds normally from the NOD/SCID mouse ing cells for up to 10.5 months. In the present experiments,we show that repopulating SRCs from CB, contained in theBM to the peripheral hematopoietic tissues.

A novel finding in this study was that B cells could de- CD34/ cell fraction, are maintained in NOD/SCID marrowand retain their capacity to home to a second marrow follow-velop in mice from a CD34-enriched fraction that had been

purged of CD19/ cells before transplanting. Whereas it could ing serial transplantation. These results suggest that theNOD/SCID model may be a useful assay for long-term re-be argued that myeloid development may have occurred

from committed progenitor cells (input fractions were con- populating cells contained in rare cell populations.taminated with low levels of CD33/ cells), this data suggests

ACKNOWLEDGMENTthat immature B cells developed de novo in the BM fromCD34/ input cells. Analysis also revealed that CD19/ cells We gratefully acknowledge the invaluable assistance of Karencomprised a comparable fraction of huCD45/ cells in mouse Helm for cell sorting and consultation on flow cytometric analysis.tissues to that found in mice transplanted with unpurged We would also like to thank Doreen Jumbeck of the National Jewish

Center Biological Resource Center for excellent care and monitoringCD34/ fractions. Furthermore, in all experiments, B cellsof experimental animals. Finally we thank Drs David Gordon andwere found to mature as they moved from the BM to theBill Wood for providing human-specific primer sequences for theperipheral circulation. While CD19/ cells in BM wereCart-1 gene in advance of publication and Dr Ralph Quinones forÉ75% to 85% negative for expression of surface IgM, thosecomments on the manuscript.found in PB were 75% to 90% positive for expression of

IgM. This shows that NOD/SCID mice are able to supportREFERENCEShuman B-cell development to the stage of cell surface Ig

1. Morrison SJ, Uchida N, Weissman IL: The biology of hemato-expression from progenitors that lack surface expression ofpoietic stem cells. Annu Rev Cell Dev Biol 11:35, 1995B lineage antigens.2. Keller G, Snodgrass R: Life span of multipotential hematopoi-Human CD14/ cells were found in BM, spleen, and PB etic stem cells in vivo. J Exp Med 171:1407, 1990for the duration of the engraftment period in all experiments 3. Jordon CT, Lemischka IR: Clonal and systemic analysis of

(up to 15 weeks). This suggests that short-term myeloid long-term hematopoiesis in the mouse. Genes Dev 4:220, 1990progenitors continued to repopulate the hematopoietic tissues 4. Trevisan M, Iscove NN: Phenotypic analysis of murine long-throughout this period of time. In vitro data also indicates term hemopoietic reconstituting cells quantitated competitively in

vivo and comparison with more advanced colony-forming progeny.that input cells able to produce CFUs were not depleted.J Exp Med 181:93, 1995Instead, evidence is presented that shows cells capable of5. Nakahata T, Ogawa M: Hemopoietic colony-forming cells ingiving rise to GM, BFU-E, and GEMM colonies in vitro,

umbilical cord blood with extensive capability to generate mono-persist after engraftment of CD34/ cells for 10 weeks. Takenand multipotential hemopoietic progenitors. J Clin Invest 70:1324,together, these data indicate that the NOD/SCID mouse pro- 1982vides an environment suitable for continuous generation of 6. McNiece IK, Stewart FM, Deacon DM, Temeles DS, Zsebo

human myeloid lineages over extended periods of time. Ad- KM, Clark SC, Quesenberry PJ: Detection of a human CFC withditionally, most myeloid cells and short-term progenitors high proliferation potential. Blood 74:609, 1989have a short half-life. Thus, the continued presence of both 7. Sutherland HJ, Eaves CJ, Eaves AC, Dragowska W, Lansdoep

PM: Characterization and partial purification of human marrow cellsmyeloid cells and colony-forming cells for extended en-capable of initiating long-term hematopoiesis in vitro. Bloodgraftment periods suggests the graft is being maintained by74:1563, 1989a primitive cell capable of extensive proliferation. Several8. Purdy MH, Hogan CJ, Hami L, McNiece I, Franklin W, Joneslines of evidence support the idea that the SRC is a primitive

RB, Bearman SI, Berenson RJ, Heimfeld S, Shpall EJ: Large volumecell with engraftment and proliferative characteristics similar ex vivo expansion of CD34 positive hematopoietic progenitor cellsto those observed in the present study.29 This report further for transplantation. J Hematother 4:515, 1995characterizes the SRC by presenting evidence that SRCs are 9. Muench MO, Roncarolo MG, Namikawa R, Barcena A, Moorecontained in the CD34/ cell population and provides a basis MA: Progress in the ex vivo expansion of hematopoietic progenitors.for analyzing highly purified CD34/ cell subfractions for a Leuk Lymphoma 16:1, 1994

10. McCune JM, Namikawa R, Kaneshima, H, Shultz LD, Lieber-more refined definition of the SRC.





HOGAN ET AL96

man M, Weissman IL: The SCID-hu mouse: Murine model for the Leif JH, Hesselton RM, Rajan TV: Improved engraftment of humanspleen cells in NOD/LtSz-scid/scid mice as compared with C.B-17-analysis of human hematolymphoid differentiation and function. Sci-

ence 241:1632, 1988 scid/scid mice. Am J Pathol 146:888, 199525. Larochelle A, Vormoor J, Lapidot T, Sher G, Furukawa T,11. Kyoizumi S, Baum CM, Kaneshima H, McCune JM, Yee EJ,

Namikawa R: Implantation and maintenance of functional human Li Q, Shultz LD, Olivieri NF, Stamatoyannopoulos G, Dick JE:Engraftment of immune-deficient mice with primitive hematopoieticbone marrow in SCID-hu mice. Blood 79:1704, 1992

12. Namikawa R, Weilbaecher KN, Haneshima H, Yee EJ, cells from b-thalassemia and sickle cell anemia patients: Implica-tions for evaluating human gene therapy protocols. Hum Mol GenetMcCune JM: Long-term human hematopoiesis in the SCID-hu

mouse. J Exp Med 172:1055, 1990 4:163, 199526. Hesselton RM, Greiner DL, Mordes JP, Rajan TV, Shultz13. Diguisto DL, Lee R, Moon J, Moss K, O’Tool T, Voytovich

A, Webster D, Mule JJ: Hematopoietic potential of cryopreserved LD: High levels of human peripheral blood mononuclear cell en-graftment and enhanced susceptibility to human immunodeficiencyand ex vivo manipulated umbilical cord blood progenitor cells evalu-

ated in vitro and in vivo. Blood 87:1261, 1996 virus type 1 infection in NOD/LtSz-scid/scid mice. J Infect Dis172:974, 199514. Kamal-Reid S, Dick JE: Engraftment of immuno-deficient

mice with human hematopoietic stem cells. Science 242:1706, 1988 27. Bock TA, Orlic D, Dunbar CE, Broxmeyer HE, Bodine DM:Improved engraftment of human hematopoietic cells in severe com-15. Nolta JA, Hanley MB, Kohn DB: Sustained human hemato-

poiesis in immunodeficient mice by cotransplantation of marrow bined immunodeficient (SCID) mice carrying human cytokine trans-genes. J Exp Med 182:2037, 1995stroma expressing human interleukin-3: Analysis of gene transduc-

tion of long-lived progenitors. Blood 83:3041, 1994 28. Lowry PA, Shultz LD, Greiner DL, Hesselton RM, KittlerLW, Tiarks CY, Rao S, Reilly J, Leif JH, Ramshaw H, Stewart FM,16. Nolta JA, Dao MA, Wells S, Smogorzewska EM, Kohn DB:

Transduction of pluripotent human hematopoietic stem cells demon- Quesenberry PJ: Improved engraftment of human cord blood stemcells in NOD/LtSz-scid/scid mice after irradiation or multiple-daystrated by clonal analysis after engraftment in immune-deficient

mice. Proc Natl Acad Sci USA 93:2414, 1996 injections into unirradiated recipients. Biol Blood Marrow Trans-plant 2:15, 199617. Turner CW, Yeager AM, Waller EK, Wingare FR, Fleming

WH: Engraftment potential of different sources of human hemato- 29. Dick JE: Normal and leukemic stem cells assayed in SCIDmice. Semin Immunol 8:197, 1996poietic progenitor cells in BNX mice. Blood 87:3237, 1996

18. Kamal-Reid S, Letarte M, Sirard C, Doedens M, Grunberger 30. Unkeless JC: Characterization of a monoclonal antibody di-rected against mouse macrophage and lymphocyte Fc receptors. JT, Fulop G, Freedman MH, Phillips RA, Dick JE: A model of human

acute lymphoblastic leukemia in immune-deficient SCID mice. Sci- Exp Med 150:580, 197931. Sutherland DR, Anderson L, Keeney M, Nayar R, Chin-Yeeence 246:1597, 1989

19. Lapidot T, Pflumio F, Doedens M, Murdoch B, Williams DE, I: The ISHAGE guidelines for CD34/ cell determination by flowcytometry. J Hematother 5:213, 1996Dick JE: Cytokine stimulation of multilineage hematopoiesis from

immature human cells engrafted in SCID mice. Science 255:1137, 32. Rowley S, Sharkis S, Hattenburg C, Sensenbrenner L: Culturefrom human bone marrow of blast progenitor cells with an extensive1992

20. Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, Ca- proliferative capacity. Blood 69:804, 198733. Gordon DF, Wagner J, Atkinson BL, Chiono M, Berry R,ceres-Cortes J, Minden M, Paterson B, Caligiuri MA, Dick JE: A

cell initiating human acute myeloid leukemia after transplantation Sikela J, Gutierrez-Hartmann A: Human Cart-1: Structural organiza-tion, chromosomal localization, and functional analysis of a carti-into SCID mice. Nature 367:645, 1994

21. Vormoor J, Lapodot T, Pflumio F, Risdon G, Patterson B, lage-specific homeodomain cDNA. DNA Cell Biol 15:531, 199634. Ng S-U, Gunning P, Eddy R, Ponte P, Leavitt J, Shows T,Broxmeyer HE, Dick JE: Immature human cord blood progenitors

engraft and proliferate to high levels in severe combined immunode- Kedes L: Evolution of the functional b-actin gene and its multi-pseudogene family: Conservation of noncoding regions and chromo-ficient mice. Blood 83:2489, 1994

22. Kollmann TR, Kim A, Zhuang X, Hachamovitch M, somal dispersion of pseudogenes. Mol Cell Biol 5:2720, 198535. Chervenick PA, Boggs DE, Marsh JC, Cartwright GE,Goldstein H: Reconstitution of SCID mice with human lymphoid

and myeloid cells after transplantation with fetal bone marrow with- Winthrobe MH: Quantitative studies of blood and bone marrowneutrophils in normal mice. Am J Physiol 215:353, 1968out the requirement for exogenous human cytokines. Proc Natl Acad

Sci USA 91:8032, 1994 36. Fulop GM, Phillips RA: The scid mutation in mice causes ageneral defect in DNA repair. Nature 347:479, 199023. Shultz LD, Schweitzer PA, Christianson SW, Gott B,

Shweitzer IB, Tennent B, McKenna S, Mobraaten L, Rajan TV, 37. Bosma MJ, Carroll AM: The SCID mouse mutant: Definition,characterization, and potential uses. Annu Rev Immunol 9:323, 1991Greiner DL, Leiter EH: Multiple defects in innate and adaptive

immunologic function in NOD/LtSz-scid mice. J Immunol 154:180, 38. Gan O, Wang JCY, Doedens M, Dick JE: The bone marrowmicroenvironment of NOD/SCID mice provides increased support1995

24. Greiner DL, Shultz LD, Yates J, Appel MC, Perdrizet G, of human hematopoiesis in vivo and in vitro. Exp Hematol 24:1118,1996Hesselton RM, Schwietzer I, Beamer WG, Shultz KL, Pelsue SC,





blood mag article

Documents