http://biotech.nature.com • MAY 2002 • VOLUME 20 • nature biotechnology

Human erythroid cells produced ex vivo at largescale differentiate into red blood cells in vivo

Thi My Anh Neildez-Nguyen1, Henri Wajcman2, Michael C. Marden3, Morad Bensidhoum4, Vincent Moncollin1,Marie-Catherine Giarratana1, Ladan Kobari1, Dominique Thierry4, and Luc Douay1,5*

New sources of red blood cells (RBCs) would improve the transfusion capacity of blood centers. Our objec-tive was to generate cells for transfusion by inducing a massive proliferation of hematopoietic stem and prog-enitor cells, followed by terminal erythroid differentiation. We describe here a procedure for amplifyinghematopoietic stem cells (HSCs) from human cord blood (CB) by the sequential application of specific combinations of growth factors in a serum-free culture medium.The procedure allowed the ex vivo expansionof CD34+ progenitor and stem cells into a pure erythroid precursor population. When injected into nonobesediabetic, severe combined immunodeficient (NOD/SCID) mice, the erythroid cells were capable of prolifera-tion and terminal differentiation into mature enucleated RBCs. The approach may eventually be useful in clinical transfusion applications.

RESEARCH ARTICLE

Erythropoiesis in adult humans results in the mean daily productionof 2 × 1011 RBCs. In medical facilities, the limited supply of humanblood products often causes shortages for patients, emphasizing theneed for new sources of RBCs. Cord blood represents a large andreadily available source of HSCs. These cells might be exploited intransfusion applications if erythroid cells could be expanded in suffi-cient numbers from primitive progenitors.

Erythropoiesis comprises the entire process by which HSCs differen-tiate in the bone marrow through several stages of precursors intomature RBCs. The process includes the commitment of pluripotentstem cells to erythroid differentiation, the erythropoietin-independentearly phase of erythropoiesis, and the erythropoietin-dependent latephase of terminal maturation of precursors into circulating enucleatedRBCs.

The use of liquid cultures of HSCs is currently limited by the pro-duction of heterogeneous mixtures of erythroid and myeloid cells1–3,or by a weak4–6 or absent terminal enucleation7,8. No procedure hasyet been developed for the production of large quantities of eithererythroid precursors or enucleated RBCs.

Many hematopoietic growth factors, including Flt3 ligand (Flt3-L), stem cell factor (SCF), and thrombopoietin (Tpo), exert positivegrowth effects on stem cells and progenitors with multilineagepotential9–11. In addition, some of these factors, such as SCF, can sup-port the differentiation of certain cell types to full maturity12.Erythropoietin (Epo) acts on committed erythroid progenitors tostimulate the later phases of erythroid differentiation13, with ery-throid colony-forming units (CFU-E) being the most Epo-sensitivecell type14,15. Insulin-like growth factor I (IGF-I) also seems to play aspecific role, as it is necessary for correct erythroid differentiation inserum-free cultures16,17.

On the basis of these observations, we designed a three-step stim-ulation protocol: first, HSC proliferation is stimulated with Flt3-L,SCF, and Tpo; second, proliferation of the erythroid progenitors

obtained in the first step is stimulated with SCF, Epo, and IGF-I; andthird, terminal erythroid differentiation is promoted with Epo andIGF-I. We show that this protocol allows CD34+ human CB progen-itor and stem cells to be massively expanded in vitro, in a serum-freemedium, into a pure erythroid precursor population. After injec-tion into NOD/SCID mice, the precursor cells proliferated furtherand achieved terminal differentiation into mature, functional, enu-cleated RBCs.

Results and discussionLarge-scale expansion of human erythroid cells. We used a cul-ture system that resulted in strong cell expansion, reaching aplateau of a 200,000-fold mean amplification of the initial cellinput (range, 50,000–280,000-fold) by day 17, with a slowing ofthe expansion rate from day 14. A decrease in cell growth related tocell mortality was observed from day 17 (Fig. 1A). Cell mortalitywas very low until day 17 (<5%) and increased considerably there-after to 70% at day 21. Erythroid differentiation was morphologi-cally evident in the differential cell count (Fig. 1B) as early as day 7(70%), whereas from day 10 onwards cultures were almost exclu-sively erythroid (93–99%). Erythropoiesis progressed from day 10,when proerythroblasts formed the predominant stage, to pro-longed culture, in which more mature cell types appeared (Fig. 1C). On day 17, 64% of the cells were basophilic erythrob-lasts, 31% were polychromatophilic or orthochromatic erythrob-lasts, and 4% were enucleated RBCs.

Characterization of the cells grown in culture. We confirmed theabove observations through immunological characterization ofthese populations (Table 1A), which showed a rapid increase in theexpression of CD36 (61 ± 15% by day 7) and a slower appearance ofglycophorin A (GPA; 68 ± 15% by day 14). Most of the cellsexpressed CD71, the transferrin receptor, throughout culture(95–99% from day 3 to 14). A decrease in expression of the transferrin

1Institut National de la Santé et de la Recherche Médicale (INSERM) U417, Hôpital Saint-Antoine, Paris, France. 2INSERM U468, Hôpital Henri Mondor, 94010Créteil, France. 3INSERM U473, Hôpital de Bicêtre, 94276 Le Kremlin-Bicêtre, France. 4Institut de Protection et de Sûreté Nucléaire, DPHD, SARAM, IPSN, 92265

Fontenay-aux-Roses, France. 5Service d’Hématologie Biologique, Hôpital Armand Trousseau, Paris, France. *Corresponding author (luc.douay@trs.ap-hop-paris.fr).

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receptor on day 17 might have accounted for the appearance of RBCs.These results were consistent with our goal of inducing terminalerythroid maturation during the third step of the culture procedure.Moreover, our procedure did not favor the growth or conservation ofother populations: <10% of the cells expressed myelomonocytic(CD14, CD15) or megakaryocytic (CD41) markers during days10–14, and no cells expressed these markers by day 17.

The chemokine receptor CXCR4 might play an important rolein the homing of early erythroid precursors to the bone marrowand in their retention in this environment18. We detected thegreatest expression of CXCR4 in the early stages of culture: thefraction of CXCR4+ cells was 60 ± 9% on day 7, 11% on day 10,and zero thereafter.

The development of a large and almost pure erythroid cell pop-ulation from day 10 of culture can probably be attributed to thepreferential proliferation of the erythroid progenitors generatedand their subsequent differentiation and maturation. Our amplifi-cation procedure indeed favored the proliferation and preserva-tion of erythroid progenitors, whereas the number of myeloidcolonies diminished rapidly. On day 10, only 1% of the cells weregranulocyte-macrophage colony-forming units (CFU-GM). Earlyerythroid burst-forming unit (BFU-E) progenitors were predomi-nant in early stages of culture but decreased rapidly thereafter.Late erythroid CFU-E progenitors proliferated extensively and byday 10 constituted 92% of the clonogenic cell population, persist-ing up to day 14, whereas the other progenitor cell types disap-peared (Table 1B).

Hemoglobin analysis. We determined the hemoglobin (Hb) con-tent of the cells to investigate the functional maturation pattern ofthe erythroid precursors in culture. Efficient Hb synthesis beginsonly at the basophilic erythroblast stage19. As expected, Hb was bare-ly detectable in the day-10 population (Fig. 2A), which was com-posed mostly of proerythroblasts (Fig. 1C). Progressive erythroidmaturation was accompanied by an increasing level of Hb: the cellshad three times more Hb on day 17 than on day 14. In both day-14and day-17 cell populations, as in the control CB sample, fetal Hb

(Hb F) was the major component (Fig. 2B). However, analysis of theγ-globin chain composition of the Hb F revealed a Gγ/Aγ ratio of60:40 in the cultured cells as compared with 70:30 in the CB sample.As this ratio is 40:60 in the trace amounts of Hb F found in theadult20, the γ-globin-chain expression of the expanded cells seems tobe between that of the newborn and of the adult. A functional studyby flash photolysis of the Hb produced by the cultured cells showedthat it bound and released O2 (data not shown). On the basis of the

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Figure 1. Large-scale expansion of human erythroid cells. (A) CD34+ hematopoietic stem cells from cord blood were grown in liquid culture according tothe three-step stimulation protocol (Experimental Protocol) and total cell numbers were counted at the indicated times. Mean values for four to sixrepresentative experiments are shown. (B) Aliquots of the cultures in A were taken at the indicated times for morphological analysis of the cells. Leuk,leukocytes; Pro/BasoE, proerythroblasts or basophilic erythroblasts; PolyE, polychromatophilic erythroblasts; OrthoE, orthochromatic erythroblasts; Retic,reticulocytes. Results are expressed as the mean percentage of the total population in three independent liquid cultures. (C) Photographs of the cells ondays 7, 10, 14, and 17 of culture. Bar, 10 µm.

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Figure 2. Analysis of the hemoglobin (Hb) produced by the expandederythroid cells. (A) Hb content, expressed as µg Hb/106 cells, wasdetermined on the indicated days. Data are mean values ± s.e.m. for threeexperiments. (B) HPLC analysis of the Hb in 14-day or 17-day expandedcells (EC). Cord blood (CB) was used as the control and the Gγ/Aγ ratio in HbF of EC or CB was measured by perfusion chromatography. One of threerepresentative experiments is shown.

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200,000-fold cell amplification achieved by day 17, we predict that astarting population of 106 CD34+ cells would produce 40–50 g of Hbin this time period.

We attempted to further differentiate the erythroid cells in vitroin the presence of various combinations of cytokines, but were notable to produce mature RBCs in large numbers. We then examineddifferentiation in vivo. Our protocol permits the amplification byday 10 of a large pool of erythroid cells comprising 93% early ery-throid precursors. The large number of CFU-E progenitors

ensures a high proliferative potential, and the expression ofCXCR4 suggests that the cells have the capacity to home to thebone marrow. We followed the evolution of these cells in vivo andassessed their ability to complete terminal maturation into func-tional enucleated RBCs.

In vivo evolution of CFSE+ human cells in NOD/SCID mice. Tofollow the in vivo fate of cells and identify cell progeny21, weinfused 30 × 106 day-10 cells, prelabeled with the intracellular flu-orescent dye carboxyfluoroscein succinimidyl ester (CFSE), intosublethally irradiated NOD/SCID mice. Interestingly, the humanerythroid cells were detectable in all organs examined (liver,spleen, bone marrow, lungs, and peripheral blood) on all days fol-lowing their injection (Figs. 3A and B). Thus, the CFSE+ humancells did not accumulate in a specific organ such as the liver orspleen where they would be eliminated, as one might have expect-ed. CFSE+ cells were detected in the peripheral circulation of themice, and their number increased markedly (from 0.5% of totalcells in peripheral blood on day 1, to 1% on day 2, to 4% on day 3,to 28% on day 4) until four days after injection and then reached aplateau (Fig. 3B). The maximum number of CFSE+ cells recordedon day 4 corresponded to an overall 96-fold amplification of thehuman erythroid cells injected on day 0, which is equivalent tonearly seven population doublings. On day 4, 28% of the totalcells in the peripheral blood of the mice were CFSE+ human ery-throid cells. This percentage was 15% on day 7. The percentage ofcirculating CFSE+ cells was determined by flow cytometry.

Terminal maturation of transfused human erythroid cells.Circulating CFSE+ cells that are negative for laser dye styryl (LDS)may represent enucleated human RBCs generated in vivo from

infused immature erythroid cells. We sorted CFSE+/LDS−

cells on day 3 for morphological analyses and on day 7 forphenotypic and biochemical studies. More than 99% of thetotal CFSE+ cells were LDS− (Fig. 3C). The day-3 and day-7CFSE+/LDS− human cells were all RBCs; thus, the circulatingCFSE+ cells represented human RBCs generated in vivo.These sorted RBCs, like the CB sample from which they werederived, expressed the human RhD blood group antigen (Fig. 4A), which appeared after expression of GPA22.

The RBCs generated in vivo synthesized mainly adult Hb(97% Hb A, 3% Hb F) with a Gγ/Aγ ratio of 35:65 (Fig. 4B), sim-

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Table 1A. FACS analyses of in vitro–expansion cultures of CD34+

cord blood cells

Positive cells (%)

Cell-surface markers Day 3 Day 7 Day 10 Day 14 Day 17Progenitors/precursorsCD71 98 ± 4 98 ± 1 95 ± 4 99 ± 2 80 ± 9ErythroidCD36 12 ± 2 61 ± 15 91 ± 3 98 ± 2 94 ± 4GPA <1 10 ± 5 38 ± 9 68 ± 15 84 ± 6Non-erythroidCD14 <0.1 3 ± 2 1 ± 1 <1 < 0.1CD41 1 ± 1 1 ± 1 <1 <1 <0.1CD15 9 ± 3 18 ± 5 9 ± 4 6 ± 4 <0.1CXCR4 ND 60 ± 9 11 ± 3 <1 <1

CD34+ hematopoietic stem cells from cord blood were grown in liquid cultureaccording to the three-step stimulating protocol (Experimental Protocol). On theindicated days, the cells were harvested and analyzed by FACS for different cellsurface markers. Results shown are means ± s.e.m. for three independentexperiments; ND, not determined.

Table 1B. Progenitor cell counts in semisolid cultures

Colonies per 104 seeded cells

Day 0 Day 3 Day 7 Day 10 Day 14 Day 17

CFU-GM 1107 ± 169 1840 ± 280 470 ± 170 15 ± 15 <1 <1BFU-E 740 ± 200 2400 ± 380 1080 ± 200 60 ± 15 <1 <1CFU-E 20 ± 20 600 ± 80 730 ± 130 851 ± 180 64 ± 7 <1

Results shown are mean ± s.e.m. for three to six independent experiments.

Figure 3. Appearance of CFSE+ human cells inNOD/SCID mice. Human cells expanded for ten dayswere labeled with CFSE before injection into the retro-orbital vein of mice (day 0). (A) FACS analyses ofCFSE+ cells in bone marrow, liver, lung, and spleen ofthe NOD/SCID mice were performed from day 1 to day7 to study the kinetics of the appearance of humancells in the various organs. Results are expressed asthe total number of CFSE+ cells in each organ on agiven day. Data shown are mean values of threeexperiments. (B) Cells from peripheral blood of theNOD/SCID mice were analyzed by FACS from day 1 today 7 to detect circulating CFSE+ human cells. Resultsare expressed as the total number of CFSE+ cells incirculation. Data shown are mean values of threeexperiments. (C) Aliquots of the cells in B obtained ondays 3 and 7 were further stained with LDS, and theCFSE+/LDS− population in peripheral blood of theNOD/SCID mice was sorted for morphologicalanalysis. Left graph, morphological gate for forwardand side scatter. Middle and right graphs, gatecorresponding to the selected CFSE+ population thatwas selected (middle) and analyzed for LDS labeling(right) and the sorting region for the CFSE+/LDS−

population (right). Photographs of sorted CFSE+/LDS−

cells (far right) were taken at two differentmagnifications; bar, 10 µm. See Experimental Protocolfor details of calculations.

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ilar to that of the fetal Hb found in adults (40:60). Thus, as comparedwith cells maturing in vitro, a switch from F cells to non-F cells hadoccurred, together with a modulation of the γ-globin chain expres-sion to the adult type. This reprogramming of the F cells to produceadult Hb suggests that complete maturation of the injected imma-ture erythroid cells may have taken place in the bone marrow of thehost mice, under the influence of an adult hematopoietic microenvi-ronment23. Hb from the sorted RBCs was able to bind and release O2

at rates similar to those of control Hb A purified from humanperipheral blood (Fig. 4C). In addition, Hb from the sorted RBCsand control Hb A exhibited comparable biphasic binding kinetics forcarbon monoxide (CO) (Fig. 4C).

Taken together, these data indicate that human erythroid prog-enitors and precursors infused into a mouse model are capable notonly of proliferating without being destroyed but also of fullymaturing into circulating RBCs. A peak level of circulating humanRBCs (28%) was observed four days after injection. More than onemonth after injection, 4% human RBCs were still present in the

peripheral blood of the mice, as determined by measure-ment of the cell surface expression of the RhD bloodgroup. Nevertheless, we feel that the murine model is notappropriate for evaluating the half-life of human RBCsgenerated in vivo because of the short half-life of endoge-nous RBCs in this model24,25 (∼ 20 days) compared withthat in humans (∼ 28 days).

Clinical relevance of the ten-day-expanded cells. Wefurther characterized our ten-day-expanded erythroidcells to assess their potential clinical value. There was noco-expansion of contaminating maternal mononuclearcells, as determined by the PCR–ASP (PCR–allele-specificprimers) method26 (data not shown); no B (CD19+) or T(CD3+) lymphocytes were detectable (<0.1%); only lowlevels of HLA class I (Fig. 5A) and HLA-DR (Fig. 5B) anti-gens were expressed by small fractions of the cells (1.4%and 3.4%, respectively); and the cells could be cryopre-served without loss of their erythroid clonogenic potential(data not shown). These characteristics support the possi-ble clinical utility of the in vitro–expanded cells for trans-fusion purposes.

We estimate that HSCs from cord blood could beexpanded as follows. About 106 CD34+ cells (correspondingto one-third to one-half of a standard CB unit banked fortransplantation) can be expanded 6,000–10,000-fold dur-ing culture for 10 days, resulting in the production of 6–10× 109 cells. These will continue to amplify in vivo aftertransfusion to give a final production of 6–10 × 1011 RBCs,equivalent to 3–5× the normal daily in vivo synthesis inhumans. Considering that one transfusable unit of packedred cells contains 1.8–2 × 1012 red cells, one cord blood unitwould provide, in these conditions, the equivalent of one tothree transfusable units of packed red cells.

CB is a rich and readily available source of HSCs. Thecryopreservation of CB ensures long-term viral security,and production of several RBC units from a single CBdonor would reduce the risks of contamination by virusesor nonconventional agents. Finally, the residual leukocytecount in the expanded product is 3–50× less than that in aclassical non-deleukocyted product.

We have described a method for the large-scale expansionof erythroid cells from cord blood HSCs. We showed that anearly erythroid precursor population generated ex vivo inlarge quantities is capable of completing terminal matura-tion in vivo, from erythroblastic precursors to mature RBCscontaining functional adult Hb. Our stem-cell technology

may find application in blood centers in conjunction with goodmanufacturing practices. Although we are not proposing to replaceclassical blood transfusion, we consider that the transfusion ofex vivo–expanded erythroid precursors may become a complemen-tary approach.

Experimental protocolCell culture. Umbilical CB units from normal full-term deliveries wereobtained with informed consent of the mothers from Saint-Vincent de PaulHospital (Paris, France). Light-density mononuclear cells were separated byFicoll–Isopaque centrifugation and enriched in CD34+ cells (typically of>90–95% purity) using Mini-MACS columns (Miltenyi Biotech, Glodbach,Germany).

The amplification procedure was based on a three-step expansion ofCD34+ cells by sequential supply of the culture with specific combinationsof cytokines27, in serum-free medium supplemented with 2% deionizedBSA, 150 µg/ml iron-saturated human transferrin, 900 µg/ml ferrous sul-fate, 90 µg/ml ferric nitrate, 100 µg/ml insulin, lipids (30 µg/ml soybeanlecithin and 7.5 µg/ml cholesterol) and 10–6 M hydrocortisone (all from

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Figure 4. Characterization of CFSE+/LDS− human RBCs sorted on day 7. (A) Sortedhuman RBCs were incubated with an anti-RhD antibody or its control IgG isotype andanalyzed by FACS. Horizontal axis, CFSE detection; vertical axis, detection of RhD orPE-conjugated control. (B) HPLC analysis of the hemoglobin (Hb) content of sortedhuman RBCs and determination by perfusion chromatography of the Gγ/Aγ ratio oftheir Hb F. (C) Functional study by flash photolysis of the Hb in sorted human RBCs.Top graph, kinetics of binding with O2 and CO after photodissociation, in samplesequilibrated under air or 1 atm CO. Rates are similar to those for control Hb A (curveswith data points). Bottom graph, normalized CO binding kinetics at differentphotolysis levels, in samples equilibrated under 0.1 atm CO. Kinetics show allostericbehavior typical of Hb A (top curve with data points). AN, normalized absorbance.

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Sigma, Saint-Quentin Fallavier, France). In the first step (days 0–7), 104/mlCD34+ cells were cultured in the presence of 50 ng/ml Flt3-L, 100 ng/mlTpo (Valbiotech, Paris, France), and 100 ng/ml SCF (R&D Systems,Abingdon, UK). In the second step (days 8–14), the cells obtained on day 7were resuspended at 5 × 104/ml in the same medium containing 50 ng/mlSCF, 3 units/ml Epo (Boehringer, Meylan, France), and 50 ng/ml IGF-I(Valbiotech). In the third step, the cells collected on day 14 were resuspended at 2 × 105/ml and cultured for a further two to seven days inthe presence of the same cytokine mixture as in the previous step but with-out SCF. The cultures were incubated at 37°C in 5% CO2 in air and themedium was changed every three days to ensure good cell proliferation.Cell differentiation was monitored throughout culture by morphologicalanalysis of the cells after cytocentrifugation and May-Grünwald-Giemsastaining. Cryopreservation of the cells on day 10 was performed indimethyl sulfoxide.

Semisolid culture assays. BFU-E, CFU-E, and CFU-GM progenitors wereassayed in methylcellulose cultures as described28. The cultures were incu-bated at 37°C in 5% CO2 in air; CFU-E colonies were counted on day 7 andBFU-E and CFU-GM colonies on day 14.

Flow cytometry. Cells (105) were labeled with unconjugated or fluoresceinisothiocyanate (FITC)-, phycoerythrin (PE)-, PerCP-, or Cy-Chrome-conjugated antibodies, washed, and resuspended in PBS containing 0.1%BSA and 1% paraformaldehyde. Antibodies to CD34, CD14, CD19 (BectonDickinson, Mountain View, CA), CD71, CD15, CD3 (Dako, Carpinteria,CA), CD41, GPA (Immunotech, Marseille, France), CD36, CXCR4(Pharmingen, San Diego, CA), RhD (clone F5; a gift from Institut National

de la Transfusion Sanguine (INTS), Paris, France),HLA class I (clone W6-32; from A. Toubert,INSERM U396), and HLA-DR (Becton Dickinson)were used for phenotyping. Controls were isotype-matched FITC-, PE-, PerCP-, and Cy-Chrome-conjugated antibodies. Analyses were performedon a FACSCalibur flow cytometer (BectonDickinson) using Cell Quest software.

NOD/SCID mouse model. All experiments and pro-cedures conformed to the French Ministry ofAgriculture regulations for animal experimentation(1987).

NOD-LtSz-scid/scid (NOD-SCID) mice wereraised under sterile conditions. Before cell injection,eight-week-old mice were sublethally irradiated with3 Gray from a 137Cs source (2.115 Gy/min). Cellsexpanded for ten days were washed, labeled withCFSE (ref. 21), and resuspended in PBS containing0.1% BSA without cytokines, then injected into theretro-orbital vein of mice (30 × 106 cells/mouse).Three animals were killed at each time point, thelungs, liver, and spleen removed, and bone marrowcells obtained by flushing the four long bones of thehind legs. Heparinized blood was drawn by retro-orbital vein puncture. Peripheral blood cells andcells extracted from the various organs were count-ed, analyzed by fluorescence-activated cell sorting(FACS) to detect CFSE+ human cells, and stainedwith the vital nucleic acid dye LDS-751 (LDS)29,30 toidentify enucleated cells (LDS− cells) in the CFSE+

population. CFSE+/LDS− cells, corresponding tohuman RBCs, were sorted from peripheral blood ondays 3 and 7 post injection. On a given day, the totalnumber of CFSE+ cells in an organ was determinedby multiplying the flow cytometric percentage ofCFSE+ cells in the organ by the total number of cells

extracted from it. The total number of CFSE+ cells in peripheral blood wasobtained by the following calculation: CFSE+ cells (%) × no. of cells/ml ofblood × blood volume (ml). The blood volume of the mice was taken to beequal to 6% of their total body weight.

Hemoglobin analyses. Hb was quantified by photometric determination at 540nm (ref. 6). The pattern of Hb synthesis was analyzed by high-performance liq-uid chromatography (HPLC, Smart system)31 on a cation-exchange Mono Scolumn (Amersham Pharmacia Biotech, Uppsala, Sweden), whereas the γ-globin composition of Hb F was determined by perfusion chromatography20.CB and peripheral blood RBCs were used as controls. The functional propertiesof the Hb produced by the expanded cells were studied by flash photolysis, asthe kinetics of ligand binding with hemoglobin tetramers exhibit a characteris-tic biphasic shape32. Ligand binding was detected at 436 nm, after photolysiswith 10-ns pulses at 532 nm.

AcknowledgmentsThe authors thank B. Drayton, M. Adam, J. Van Nifterik, A. Yapo, R. Gilot, andthe cytometry team of Armand Trousseau Hospital for technical assistance, P.Gane for the gift of RhD antibodies, and Y. Brossard, P. Rouyer-Fessard, and Y.Bezard for helpful discussions. We are also grateful to M. Ardouin for the valuable gift of NOD/SCID mice. This work was supported by grants from theAssociation pour la Recherche en Transfusion, the Etablissement Français desGreffes, and La Ligue Contre le Cancer. T.M.A.N.-N. received a grant from theAssociation Combattre la Leucémie.

Received 28 August 2001; accepted 14 February 2002

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Figure 5. HLA class I and HLA-DR analyses of cells expanded for ten days. (A) Two-colorfluorescence dot plots of HLA class I and CD36 antibodies and their respective FITC- and PE-conjugated isotype controls. (B) Two-color fluorescence dot plots of HLA-DR and CD36 antibodiesand their respective FITC- and PE-conjugated isotype controls.

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