Ex vivo expansion of human umbilical cord blood and peripheral blood CD34+ hematopoietic stem cells

Experimental Hematology 28 (2000) 1297–1305

Ex vivo expansion of human umbilical cord blood and peripheral blood CD34⫹ hematopoietic stem cells Gary L. Gilmore, Darlene K. DePasquale, John Lister, and Richard K. Shadduck The Western Pennsylvania Cancer Institute, Pittsburgh, Pa., USA (Received 21 January 2000; revised 21 July 2000; accepted 25 July 2000)

Objective. The proliferation and expansion of human hematopoietic stem cells (HSC) in ex vivo culture was examined with the goal of generating a suitable clinical protocol for expanding HSC for patient transplantation. Materials and Methods. HSC were derived from umbilical cord blood (UCB) and adult patient peripheral blood stem cell collections. HSC were stimulated to proliferate ex vivo by a combination of two growth factors, flt-3 ligand (FL) and thrombopoietin/c-mpl ligand (TPO/ML), and assessed for expansion by flow cytometry. Results. Ex vivo expansion cultures of UCB were maintained for prolonged periods (up to 16 weeks), and sufficient HSC were generated for adult transplantation. In contrast to UCB, FL ⫹ TPO/ML did not significantly increase CD34⫹ peripheral blood stem cell (PBSC) numbers. Conclusion. UCB-HSC can be expanded in culture to numbers theoretically adequate for safe, rapid engraftment of adult patients. Additional studies are needed to establish the functional activity of expanded UCB-HSC. © 2000 International Society for Experimental Hematology. Published by Elsevier Science Inc. Keywords: Stem cells—Ex vivo expansion—Umbilical cord blood—Peripheral blood

Introduction Umbilical cord blood (UCB), collected from the postpartum placenta, contains hematopoietic stem cells (HSC) that can serve as an alternate source to bone marrow (BM) or peripheral blood stem cell (PBSC) preparations for HSC transplantation [1]. UCB has been used successfully for over 800 pediatric transplants [2], but both adult and pediatric patients receiving UCB transplants have engrafted more slowly than those receiving either BM or PBSC grafts [3], the delay being more severe in adult patients. The rate of engraftment is dependent on the number of infused stem cells, and the CD34⫹ content of UCB is, at most, 5% of the optimal dose for adults (2–4 ⫻ 106 CD34⫹/kg). A reproducible method of expanding the HSC population is needed for UCB to become a safe and reliable resource for adult HSC transplantation. Previous work on the ex vivo expansion of human HSC populations examined human adult stem cells (BM or PBSC) as input [4–11]. These cultures used a complex mixture of cytokines at high concentrations, and achieved only limited expansion of CD34⫹ cells (twofold–10-fold). MoreOffprint requests to: Gary L. Gilmore, Ph.D., Suite 2303 NT, The Western Pennsylvania Hospital, 4800 Friendship Avenue, Pittsburgh, PA 15224-2207 USA; E-mail: [email protected]

over, there was no evidence of expansion of long-term culture-initiating cells (LTC-IC), and cultures could be maintained for at most two weeks. A recent study examined the effect of a simpler culture medium, with two cytokines, flt-3 ligand (FL) and thrombopoietin/c-mpl ligand (TPO/ML), on the expansion of UCB stem cell populations [12]. The authors demonstrated significant expansion of HSC populations, including LTC-IC, that could be maintained longterm (up to six months). The initial report, which employed only FL and TPO/ML as growth factors, showed expansion of lymphoid as well as myeloid progenitors, suggesting these cultures contained true pluripotent HSC. The present studies tested the feasibility of growing sufficient HSC from a single cord blood collection for adult transplantation and assessed whether FL ⫹ TPO/ML could stimulate expansion of adult HSC populations. A series of cultures were initiated with CD34⫹ cells purified from UCB or PBSC. FL ⫹ TPO/ML stimulated expansion of CD34⫹ cells from UCB, using either purified CD34⫹ cells or whole UCB mononuclear cells. CD34⫹ PBSC purified from adult patients, on the other hand, were not stimulated to proliferate by FL ⫹ TPO/ML to any significant extent; furthermore, CD34 content of these cultures decayed rapidly after 14 days, followed by a rapid loss in cellularity. Addition of supernatant from actively proliferating cord blood cultures

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(UCB-SN) stimulated a marginal proliferation of CD34⫹ PBSC. However, expansion was transient and was followed by a decline in CD34⫹ cell numbers, as in the cultures with FL ⫹ TPO/ML alone. If such a putative “expansion factor” could be identified and purified, this might permit generation of large numbers of HSC from limited numbers of input PBSC, and could improve the safety of HSC transplantation.

Cytokines Recombinant human flt-3 ligand (FL) was the kind gift of Dr. Douglas Williams (Immunex, Seattle, WA). Two forms of c-mpl ligand (ML) were tested; recombinant human thrombopoietin (TPO) was donated by Genentech (San Francisco, CA) and recombinant human megakaryocyte growth and differentiation factor (MGDF) was the gift of Dr. Janet Nichols (Amgen, Thousand Oaks, CA), who also supplied recombinant human stem cell factor (SCF, also known as mast cell factor [MGF] or c-kit ligand [KL]). Natural human interleukin-6 was the generous gift of Honora Cooper Eckhardt (Novartis, Summit, NJ).

Materials and methods UCB and patient PBSC samples UCB was collected after obtaining consent from patients scheduled to undergo Cesarean section. On average, 60 mL of UCB was collected by gravity into a sterile container after cutting the distal end of the cord. Heparin (1000 U) was added to prevent coagulation. White blood cell (WBC) counts were measured with a Coulter counter (Beckman/Coulter, Miami, FL). The sample was diluted with an equal volume of Hank’s balanced salt solution (HBSS) (Sigma, St. Louis, MO), and layered onto Ficoll-Paque (Pharmacia-Amersham, Piscataway, NJ; ␳ ⫽ 1.077 g/mL) density gradients to deplete red blood cells (RBC). The mononuclear cell interface was collected, diluted into three volumes of HBSS, and pelleted at 250 ⫻ g for 10 minutes. The cell pellet was washed two more times and resuspended in either 5 mL of expansion media (for culture of RBC-depleted cord blood cells) or 10 mL Isolex working buffer (Dulbecco’s phosphate-buffered saline [D-PBS] at pH ⫽ 7.4/1% detoxified human serum albumin [HAS]/0.4% sodium citrate) for CD34⫹ cell purification. When necessary, RBCdepleted UCB cells were stored frozen at ⫺70⬚C after suspending the cells in HBSS at a concentration of ⱕ 2 ⫻ 108 cells/mL, and adding an equal volume of pentastarch cryopreservative medium [13] containing 10% dimethyl sulfoxide (DMSO)/8% HSA/12% pentastarch in normosol R, at a final cell concentration ⱕ 108 cells/ mL. For further processing, frozen samples were thawed in a 37⬚C water bath. CD34⫹ peripheral blood stem cell (PBSC) samples were obtained (with informed consent) from the Blood and Marrow Processing Laboratory of the Western Pennsylvania Cancer Institute. Patients were treated with cyclophosphamide (4 g/m2) and either etoposide (600 mg/m2) or paclitaxel (170 mg/m2) followed by G-CSF (Amgen, Thousand Oaks, CA) (5–8 ␮g/kg) subcutaneously every 12 hours. PBSC were collected after the leukocyte count exceeded 1000 cells/␮l, or when the absolute CD34⫹ count exceeded 50 cells/␮l. CD34⫹ PBSC were isolated from leukopheresis products by magnetic bead separation with the Isolex 300i device (Nexell, Irvine, CA). Purification of CD34⫹ cells from UCB CD34⫹ cord blood cells were isolated using the Isolex 50 device, according to the manufacturer’s instructions. Human IgG (Gamma-Gard; Baxter, Irvine, CA) was used at 2 mg/mL to block nonspecific binding. Mouse monoclonal antibody to human CD34 (clone 9C5) was used at 50 ␮g per 108 cells, and magnetic beads coupled to anti-mouse antibody were added at 0.5 beads/cell. The selected CD34⫹ cells were released from the beads using a nonenzymatic releasing agent (a peptide “mimitope” of CD34). The selected cells were washed, counted, and sampled for CD34 analysis to determine yield and purity.

Ex vivo expansion cultures Expansion culture media (Iscove’s modified Dulbecco’s medium\10% FCS\50 ␮g/mL gentamicin sulfate\50 ng/mL FL\35 ng/ mL ML [TPO or MGDF]) was prepared fresh weekly. CD34⫹ PBSC (0.5–2 ⫻ 106 total cells), RBC-depleted UCB (1–10 ⫻ 107 total cells), or CD34⫹ UCB (0.1–4 ⫻ 106 total cells) were incubated in 25 cm2 tissue culture flasks in 3–5 mL expansion media in a fully humidified atmosphere of 5% CO2\5% O2 at 37⬚C. Cultures were fed by addition of one-half volume fresh expansion media twice a week. Following vigorous pipetting to suspend all but a small number of tightly adherent cells, one-half of the culture was removed at weekly intervals for CD34 analysis, subculture, or frozen storage. Total cell counts were taken at each feeding. Cells from cultures with low cell numbers or slow growing cultures would be fed back in one-half volume fresh expansion media. For subculture, harvest, or transfer, adherent cells were either detached by cell scraper or released by trypsin/EDTA treatment followed by a wash in IMDM ⫹ 10% FCS and resuspended with the nonadherent fraction. Supernatants were collected from UCB cultures showing significant expansion of CD34⫹ cells to test for enhancing expansion of adult CD34⫹ PBSC (see below). Flow cytometry analysis of CD34⫹ populations from expansion cultures Initially, samples were analyzed by two-color flow cytometry on a FACScan or FACScaliber analyzer (Becton-Dickinson, San Jose, CA); recent samples have been analyzed using either a Coulter XL analyzer or Altra cell sorter (Beckman-Coulter Corp., Miami, FL). 0.1–1 ⫻ 106 cells were stained with FITC-conjugated anti-human CD45 and PE-conjugated anti-human CD34, and gated for CD45⫹ CD34⫹ cells with low side scatter, according to the CD34 enumeration protocols developed by the International Society of Hematotherapy and Graft Engineering (ISHAGE) [14]. A replicate sample was stained with FITC–anti-CD45 and PE-mouse IgG1 as an isotype control to ensure specificity.

Results Isolation of CD34⫹ cord blood cells Over 130 cord blood samples have been collected. The average sample volume was 57 mL (range: 9–112 mL), containing on average 1.26 ⫻ 107 WBC/mL (range: 5.24 ⫻ 106–3.63 ⫻ 107). The average frequency of CD34⫹ cells in whole cord blood was 0.41% by flow cytometry (range: 0.05–1.21%). Samples containing ⬎ 2 ⫻ 108 total WBC (n ⫽ 100) were centrifuged over Ficoll-Paque (Pharmacia-Amer-

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sham) to deplete erythrocytes. Mean recovery of MNC was 35% of input (range: 16–52%) and 70% of CD34⫹ cells (range: 32–99%). This twofold enrichment of CD34⫹ cells has been noted by other groups [15–17]. Unlike either peripheral blood or bone marrow, cord blood mononuclear cell preparations contain nucleated red cells that are resistant to hypotonic lysis. CD34⫹ cells were isolated from preps with ⱖ1 ⫻ 108 mononuclear cells after Ficoll-Paque (n ⫽ 93). The average yield of MNC was 0.85% (range: 0.5–1.6%); the average purity was 63.9% (range: 36–99%), and the average recovery of CD34⫹ cells was 55% of the starting material (range: 23–99%). Flow cytometric analysis of CD34⫹ cells in the CD34-depleted fraction revealed that these cells had a lower mean fluorescence intensity (MFI) than did the CD34selected population (215 vs 1258). This suggests these cells express CD34 at insufficient levels to be captured by the magnetic bead separation technique. The average number of CD34⫹ cells recovered per 1 ⫻ 108 WBC input was 2.27 ⫻ 105 when fresh cord blood was processed, and 2.03 ⫻ 105/ 108 WBC when frozen cord blood was used. Thus, frozen samples are suitable for this procedure, as the yields from frozen cord blood samples are ⱖ90% of the yields from fresh cord blood samples. Expansion of cord blood CD34⫹ cells Samples of RBC-depleted UCB (1–4 ⫻ 107 cells) or CD34⫹ UCB (0.3–5 ⫻ 106 cells) were cultured in expansion media

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as described. Figure 1 shows the cumulative production of CD34⫹ cells (as percent of input) across time for CD34⫹ cultures (data from the first 30 cultures). CD34⫹ cord blood expansion cultures can be maintained up to 19 weeks; the average time in culture was 8.9 weeks (⫾2.1). The mean cumulative expansion of total MNC was 1385 times input, and the mean cumulative expansion of CD34⫹ cells was 89.9 times input. Thus, an average cord blood prep containing 1.1 ⫻ 106 CD34⫹ cells (1.5 ⫻ 106 total cells) produces on the order of 1.0 ⫻ 108 CD34⫹ cells (2.1 ⫻ 109 total cells) after 9 weeks in culture (1.3 ⫻ 106 CD34⫹ cells/kg for a 75 kg patient). A similar overall expansion of MNC and CD34⫹ cells is seen when RBC-depleted UCB is used as input instead of CD34⫹ selected UCB (data not shown). During these cultures, the percentage of CD34⫹ cells increased from 0.45% to 3.0%, as opposed to the decrease that occurred in CD34⫹ cultures. However, there was a 4- to 5-week lag before CD34⫹ expansion occurred, indicating that the stem cell proliferation may have been inhibited by mature lymphocytes, granulocytes, and monocytes present in the unseparated cultures. Kinetics of CD34⫹ cell expansion Kinetic analysis of total CD34⫹ cell production during expansion shows a lag of 6 to 10 days, followed by a 14- to 21-day period of exponential growth; then the rate of expansion slows dramatically (Fig. 2). This result suggests two

Figure 1. Expansion of purified CD34⫹ umbilical cord blood cells (UCB). CD34⫹ UCB grown in flt-3 ligand (FL) ⫹ thrombopoietin/c-mpl ligand (TPO). Expansion is plotted as % of input (logarithmic scale) vs days in culture. (Data from 30 experiments)

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Figure 2. Kinetics of CD34⫹ expansion. Average fold expansion is plotted vs time in culture (linear scale). Note three phases of expansion: a) limited initial expansion; b) rapid expansion; and c) slower late expansion.

testable hypotheses about stem cell expansion: 1) the initial lag is caused by a need for additional hematopoietic growth factors (beyond FL and TPO/ML) that must accumulate to sufficient concentration before exponential growth can occur, and 2) CD34⫺ cells produced during culture inhibit CD34⫹ expansion, either by consuming the growth factors or by producing hematopoietic inhibitors, such as macrophage inflammatory protein-1␣ (MIP-1␣), tumor necrosis factor-␣, tumor growth factor-␤ [18], or human monokine induced by interferon-␥ (HuMIG) [19]. To test the hypothesis that additional cytokines are required for exponential stem cell expansion, two approaches were taken. First, supernatant (UCB-SN) from 2- to 3-week expansion cultures was added to freshly isolated CD34⫹ UCB to a final concentration of 20% in fresh expansion medium. No growth was seen in any of four cultures initiated with 20% UCB-SN (data not shown), suggesting that 20% UCB-SN contains insufficient cytokine(s) to stimulate exponential expansion, or that UCB-SN contains hematopoietic inhibitors. The second approach was to add exogenous human stem cell factor (SCF) and interleukin-6 (IL-6) to the expansion medium at concentrations of 50 ng/mL and 10 ng/mL, respectively, as suggested by Dr. W. Piacibello (personal communication). Cultures containing the four growth factors doubled their CD34⫹ content by day 5, while little, if any, expansion was seen in standard cultures with FL ⫹ TPO/ML at this time point (data not shown). This result shows that adding SCF ⫹ IL-6 accelerates stem cell expansion early in culture, indicating that these factors are needed for optimal stem cell expansion. UCB HSC can be grown long-term in FL ⫹ TPO/ML ⫹ SCF ⫹ IL-6 (16

weeks), with greater CD34⫹ yields than with FL ⫹ TPO/ ML expansion medium. Four-factor medium is now the standard expansion culture system of the laboratory. To test the hypothesis that mature cells inhibit expansion of CD34⫹ cells, CD34⫹ cells were reisolated from threeweek-old cultures and replated in standard expansion medium; parallel cultures were established with equivalent numbers of CD34⫹ cells without reisolation, and CD34⫹ production was compared. Figure 3 shows that reisolation of CD34⫹ cells restores exponential growth potential, while expansion in the parallel cultures is slower and linear, as would be expected from the kinetics of CD34⫹ expansion. This result demonstrates that CD34⫺ cells produced during culture inhibit the rate of stem cell expansion. These experiments cannot distinguish between consumption of growth factors and production of hematopoietic inhibitors, although the results from adding UCB-SN to fresh CD34⫹ UCB cultures imply the existence of inhibitory activity in expansion culture supernatants. Expansion of CD34⫹ cord blood stem cells in serum-free medium Stem cell expansion in serum-free conditions was examined by splitting established cultures in expansion medium where the FCS was replaced by bovine serum albumin (BSA), bovine insulin, and human transferrin. Growth of cultures split by dilution into serum-free expansion media was indistinguishable from replicate cultures grown in FCScontaining media (data not shown). We also tested commercially produced serum-free media, QBSF-60 (Quality Biologicals, Gaithersburg, MD). This medium contains detoxified

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Figure 3. Reisolation of CD34⫹ cells restores rapid expansion of UCB cultures. Parallel cultures established with identical input # of CD34⫹ cells; either reisolated (solid line; 䊐) or subcultured to equivalent # of CD34⫹ cells (dashed line; 䉬) at times indicated by arrows ( ↓).

human serum albumin, human transferrin, and recombinant human insulin, and is produced according to certified good laboratory practice (cGLP) standards. Expansion of CD34⫹ cells was enhanced in these cultures (three- to fourfold)

when compared to replicate FCS-containing cultures, even though total MNC increases were similar (Fig. 4). Three cultures of CD34⫹ CB were initiated in QBSF-60 expansion medium; surprisingly, none showed expansion of

Figure 4. Enhanced CD34⫹ expansion in QBSF-60 serum-free medium. Established expansion cultures were split into standard FL ⫹ TPO medium containing FCS (std; solid bars) or commercial QBSF-60 medium with FL ⫹ TPO (QBSF; open bars). (Mean of four experiments)

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Figure 5. CD34⫹ UCB expansion fails to initiate without serum. Growth curves of equivalent # of CD34⫹ UCB from same collection seeded into std (䉬) or QBSF expansion medium (o); QBSF culture fails to grow until switched into std medium ( ↓).

CD34⫹ cells, or even MNC. Concurrent cultures set up in standard expansion medium showed enhanced expansion of CD34⫹ cells when split into the same batch of QBSF-60, indicating that there was no problem with the medium. It was unclear why this occurred, but one difference between the serum-free and FCS-based cultures was the absence of adherent cells in the serum-free cultures. Potentially, serum is needed to permit attachment of feeder cells, which supports stem cell expansion, and once that has occurred, serum is no longer necessary. This deduction was substantiated by an experiment where a freshly isolated high-yield UCB preparation was divided into two flasks: one with QBSF-60 expansion medium and one with standard FCS-containing medium. The growth curve is shown in Figure 5. For two weeks, there was no growth in the QBSF-60 flask, while the standard culture increased 19.7-fold, with a 6.2-fold CD34⫹ cell expansion. CD34⫹ expansion was triggered by switching the cells from serum-free medium to FCS containing medium (indicated by the arrow). Since serum is required for initiation of expansion conditions, we examined the ability of cord blood plasma to substitute for FCS, reasoning that autologous plasma should contain the requisite factor(s). Cord blood plasma (CBP)expansion medium was prepared, and new and established cultures were grown in CBP-based media. When sufficient numbers of CD34⫹ cells were isolated from a single preparation to permit replicate cultures, simultaneous cultures were established in CBP-based and FCS-based expansion media. In all cases, CBP was at least equivalent, if not superior, to FCS in supporting the expansion of CD34⫹ cells, in

both new and established cultures (data not shown). Thus, CBP has the required serum activity and may be used in place of FCS. Absence of expansion of adult CD34⫹ PBSC In contrast to CD34⫹ UCB, CD34⫹ PBSC from patients show limited, if any, expansion when cultured with FL ⫹ TPO/ML. Samples of CD34⫹ PBSC from 70 patients were obtained from the Blood and Marrow Processing Laboratory of the Western Pennsylvania Cancer Institute. The types of malignancy are shown in Table 1. The initial 10 samples were cultured in 5 mL of media with FL ⫹ TPO/ ML at a cell density of 0.4–5 ⫻ 106 cells/mL the results are shown in Figure 6. Though total cellularity increased threeto fourfold in the first two weeks, expansion of CD34⫹ cells was limited (115–150% of input), then cell production declined rapidly. CD34 levels diminished to ⬍1%, and cultures became static by the fourth or fifth week. Addition of SCF ⫹ IL-6 to the expansion medium did not improve CD34⫹ PBSC expansion (data not shown). Table 1. CD34⫹ PBSC Samples: Patient Malignancies Diagnosis Breast Cancer Non-Hodgkin’s Lymphoma Multiple Myeloma Hodgkin’s Disease Ovarian Carcinoma Acute Myeloid Leukemia Total # of Patients

# of Patients

% of Total

27 21 12 5 3 2 70

39% 30% 17% 7% 4% 3%

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Figure 6. CD34⫹ peripheral blood stem cells (PBSC) fail to expand in FL ⫹ TPO medium. CD34⫹ PBSC grown in std FL ⫹ TPO medium, with minimal evidence of expansion. Decay of CD34⫹ PBSC is plotted as % of input vs days in culture. (Data from 16 experiments)

UCB-SN was added to 20 different CD34⫹ PBSC cultures (20% final concentration). UCB-SN appeared to contain an activity that augmented adult HSC expansion; addition of SN enhanced both total cellular proliferation (fourfold to 11-fold) and stem cell expansion (150–200%). This is the opposite result from CD34⫹ UCB cultures, where UCB-SN inhibited growth. Stem cell expansion could not be maintained; CD34⫹ levels subsequently fell to ⬍1% of input by week 4. Total cell output was sustained for three to five weeks, then decayed, paralleling the decline in CD34⫹ cells. These results are far from striking, but they support the hypothesis that UCB expansion cultures produce an activity that stimulates expansion of adult HSC. It is possible, from the results with serum-free expansion medium on established UCB cultures, that FCS inhibits adult HSC expansion; however, there was no improvement in CD34⫹ PBSC expansion either in QBSF-60 based expansion medium or when supplemented with UCB-SN generated in serum-free medium (data not shown).

Discussion The ability to expand human CD34⫹ stem cell populations from normal umbilical cord blood and patient peripheral blood has been examined, with a focus on eventual clinical application. The results of this study clearly show the feasibility of the technique, in that one million CD34⫹ UCB cells (or less) can be expanded to one hundred million CD34⫹ cells (or more) in eight to nine weeks. Both fresh and cryopreserved cord blood collections can be expanded equally

well in culture. This would permit use of the growing pool of banked cord blood collections for adult allogeneic transplant patients. Ex vivo expanded UCB HSC might shorten the period of aplasia seen with fresh UCB transplants. The clinical benefit is obvious. Furthermore, aliquots of expanded cells could be stored frozen to serve as “seed” cultures, so the expanded cord blood preparation might be available for several recipients. Another important aspect of expansion culture is the success rate, the reproducibility of expansion. Of 60 cultures established with the standard FL ⫹ TPO/ML medium, 57 demonstrated significant expansion of CD34⫹ HSC. It is interesting to note that all cultures that failed were seeded with less than 2.2 ⫻ 105 total cells, and only two cultures seeded with less than 2.2 ⫻ 105 total cells expanded. Both of these cultures were ⬎85% pure CD34⫹ cells. This indicates there may be a minimum number of purified stem cells necessary to establish a productive culture in a 25 cm2 tissue culture flask. This low failure rate (5%) means the chance for successful stem cell expansion of any given cord blood sample is excellent, which is essential for patient therapy. A high failure rate would preclude clinical application of this technology. It is also clear that success in expanding CD34⫹ UCB is dependent on isolation of the CD34⫹ population, even though expansion does occur in RBC-depleted UCB cultures. Omission of the CD34 isolation would both simplify and reduce the cost of the procedure, but the resulting delay (five weeks or more) seems unacceptable for good management of patients with hematological malignancy. Although the success rate was high, the expansion tech-

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nique still requires optimization at several points. Collecting larger volumes of cord blood (e.g., by cannulating the umbilical vein) should increase the total stem cell yield, but also increases the number of density gradients needed to process the preparation, thereby increasing overall cell loss. Use of a large-volume chamber for erythrocyte depletion, such as those produced by Fresenius (Taunusstein, Germany), might improve yields, because the cells are transferred a single time between the gradient and collection chambers. Modification of the magnetic selection procedure may improve yield, in that as much as 25% of the input CD34⫹ population is recovered in the CD34-depleted fraction. These cells express CD34 at lower levels than the CD34selected population, based on mean fluorescence intensity. It is unclear what alterations will enable capture of these cells. A second human stem cell antigen has been defined by the monoclonal antibody AC133 [20], and it is possible that a combination of CD34 and AC133 might permit isolation of high-purity stem cell populations, which could result in enhanced expansion in this system. There is a high degree of variability in the early stages of expansion (mean ⫽ 6.7-fold [⫾4.4] at 2 weeks). We have analyzed this data with respect to fresh vs frozen cord blood, purity, and initial plating density, and have found no statistically significant variable. The basis of this variation is unclear but is not unexpected in normal donor samples. We and others have found that adding other hematopoietic growth factors, particularly human stem cell factor (SCF), to the cultures increases expansion of UCB stem cells, decreasing the time needed to generate a sufficient number of stem cells for rapid engraftment of adults. An additional class of cytokines that may be particularly useful for improving stem cell expansion are certain hematopoietic inhibitors, such as leukemia inhibitory factor (LIF), or MIP1␣, which has been reported to inhibit replication of a variety of hematopoietic progenitors, but not LTC-IC [21]. This needs to be approached systematically and carefully to be sure that the added factors do not enhance stem cell differentiation or depletion. We have found CD34⫹ expansion is enhanced (up to fourfold) with commercial serum-free medium, QBSF-60. The benefits of using defined serum-free medium are absence of FCS and other bovine-derived components, which obviates concern over the potential presence of allergens or infectious agents, like bovine spongiform encephalitis (BSE) or other prion-type diseases, which could be transmitted to the patient. The faster stem cell expansion in QBSF-60 could decrease the total volume of expansion medium needed to produce a cord blood graft with sufficient HSC for adult transplantation, thereby decreasing the cost of the procedure. The basis of enhanced expansion cells is unknown. One possibility is that FCS may contain either hematopoietic inhibitors (TNF-␣) or differentiation factors (GM-CSF, IL-3) that stimulate hematopoiesis in vitro,

thereby depleting the CD34⫹ population. QBSF-60 medium is produced with defined human components (detoxified serum albumin, recombinant insulin, and detoxified transferrin) and lacks these activities. Our results with CD34⫹ PBSC reflect findings from other ex vivo expansion systems; HSC from adult sources apparently have limited potential to expand in vitro, even in media containing FL ⫹ TPO/ML. This may be either due to a qualitative difference between adult and neonatal stem cells, as suggested by results with NOD/SCID mice [22], or because CD34⫹ UCB contain ancillary cells that provide support for stem cell expansion that CD34⫹ PBSC lack, as has been shown in bone marrow cultures [23]. In support of the second hypothesis, supernatant from actively proliferating cord blood cultures (UCB-SN) transiently stimulated a marginal proliferation of CD34⫹ PBSC. This suggests that there may be a factor or factors present in UCB-SN that stimulate proliferation of adult CD34⫹ PBSC, although this activity appears to be in limiting amount. This is a difficult assay to standardize, both in terms of input PBSC and UCB-SN quality, making this research somewhat speculative. However, the potential benefit of this putative “expansion factor” is significant. If it can be identified as either a known or a novel cytokine, it could be used to generate large numbers of HSC from limited numbers of input PBSC. This could improve the safety of HSC transplantation and reduce the cost and stress of repeated leukophereses to the patient, thereby benefiting a large number of cancer patients. It is important to determine whether our expanded stem cells function normally, that they can engraft and yield normal blood cell production following extended culture. This can be tested in vivo using NOD/SCID recipients, which accept human stem cell grafts after receiving a low dose of irradiation. We have recently begun investigating this area, and our preliminary findings support the hypothesis that expanded UCB HSC retain stem cell function, which concurs with a recently published study [24]. Thus, the goal of developing a clinically suitable method for expanding cord blood stem cells ex vivo appears feasible and should be of significant benefit to patients who need an allogeneic transplant but lack a related matched stem cell donor. Acknowledgments The authors would like to thank the Labor and Delivery Staff of the Western Pennsylvania Hospital for their assistance in collecting cord blood samples. This work was supported by funds provided by the Western Pennsylvania Hospital.

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