Stem Cell Biology Laboratory, Large Scale Biology Corporation, Vacaville, California, USA;

Transcription

1 Stem Cells Original Article Quantitative Analysis Demonstrates Expansion of SCID-Repopulating Cells and Increased Engraftment Capacity in Human Cord Blood Following Ex Vivo Culture with Human Brain Endothelial Cells JOHN P. CHUTE, a,b GARRETT MURAMOTO, a JENNIFER FUNG, c CAROL OXFORD d a Stem Cell Biology Laboratory, Large Scale Biology Corporation, Vacaville, California, USA; b Division of Hematology/Oncology, University of California at Davis Cancer Center, Sacramento, California, USA; c Jackson Laboratories, Sacramento, California, USA; d University of California at Davis Medical Center, Department of Pathology, Davis, California, USA Key Words. Cord blood SCID-repopulating cells Ex vivo expansion Endothelial cells ABSTRACT Initial clinical trials examining the transplantation of ex vivo expanded cord blood (CB) cells have failed to demonstrate an impact on hematopoietic recovery compared with historical unmanipulated CB controls. In this study, we tested whether coculture with primary human brain endothelial cells (HUBECs) could increase the engraftment capacity and repopulating cell frequency within CB CD34 + cells. Quantitative analysis demonstrated that HUBEC coculture for 7 days supported a 19-fold greater number of CD34 + cells and 3.4-fold and 2.6-fold greater severe combined immunodeficient (SCID)-repopulating cell (SRC) frequencies than fresh CB CD34 + cells and liquid suspension-cultured cells. Mice transplanted with day-14 HUBEC-cultured cells showed 4.2-fold higher levels of human engraftment than mice transplanted with day-7 HUBEC-cultured cells, indicating that SRC enrichment continued to occur through day 14. Noncontact HUBEC cultures also maintained SRCs at levels comparable with contact HUBEC cultures, demonstrating that HUBECsecreted soluble factors critically supported SRC selfrenewal. Seeding efficiency studies demonstrated that HUBEC-cultured CB CD34 + cells engrafted nonobese diabetic/scid marrow at significantly higher levels than either fresh CB CD34 + cells or liquid suspension-cultured CD34 + cells. These studies indicate that the application of HUBEC coculture or HUBEC-conditioned media can potentially improve upon current strategies for the clinical expansion of CB stem cells. Stem Cells 2004;22: INTRODUCTION Umbilical cord blood (CB) is an attractive source of hematopoietic stem cells for transplantation due to its high concentration of stem/progenitor cells [1], lower risk for graft-versus-host disease [2, 3], and relative availability and ease of procurement [4, 5]. However, clinical application of CB for transplantation has been limited by the low absolute stem cell numbers per graft [6-8], risk of graft failure [9-11], and slower platelet and neutrophil engraftment compared with other sources of adult stem cells [2-4]. A rationale has, therefore, existed to attempt ex vivo expansion of CB stem cells for application in larger-sized children and adults Correspondence: John P. Chute, M.D., Stem Cell Transplantation Program, Duke University, 2400 Pratt Street, Suite 1100, Durham, North Carolina 27710, USA. Telephone: ; Fax: ; johnchute@duke.edu Received September 5, 2003; accepted for publication October 28, AlphaMed Press /2004/$12.00/0 STEM CELLS 2004;22:

2 Chute, Muramoto, Fung et al. 203 [4, 12] and to accelerate the hematopoietic and immune system recovery following transplantation [13]. In the steady state, the CD34 + CD38 population is enriched for primitive hematopoietic cells capable of repopulating nonobese diabetic severe combined immunodeficient (NOD/SCID) mice (SCID-repopulating cells [SRCs]) [14]. However, following ex vivo culture, dissociation between CD34 + CD38 cell expansion and SCID-repopulating capacity has been observed [15]. Therefore, investigators have focused on the SCID-repopulating capacity of cultured hematopoietic cells rather than the phenotype as a measure of stem cell frequency [16]. Recent preclinical studies have indicated that human CB CD34 + cells can be expanded in vitro under various conditions for up to 1-10 weeks [17-20]. Liquid suspension culture conditions, including stem cell factor (SCF), Flt-3 ligand, and thrombopoietin (TPO), have also been reported to optimize the ex vivo maintenance of SRCs within human CB [19-21]. However, a recent study suggested that ex vivo expansion of human CB stem cells in stroma-free conditions may not maintain nor expand long-term repopulating cells as effectively as previously hypothesized [22]. That study demonstrated that CB stem cells cultivated in vitro with megakaryocyte growth and development factor (MGDF) + SCF + G-CSF were unable to provide long-term repopulation in a fetal sheep model [22]. As importantly, those investigators also demonstrated, in a phase I clinical trial, that transplantation of unrelated donor CB CD34 + cells, which were cultured for 10 days with SCF + G-CSF + MGDF, did not result in faster in vivo hematopoietic recovery as compared with historical unmanipulated CB transplant controls [23]. Jaroscak et al. also showed that infusion of ex vivo expanded CB cells cultured under stroma-free conditions with PIXY 321 (GM-CSF-interleukin [IL]-3 fusion protein) + Flt-3 ligand + erythropoietin did not impart a faster recovery of either platelets or neutrophils compared with historical controls [13]. The hematopoietic capacity of endothelial cells has been suggested by the interdependent development of vascular endothelial precursors and hematopoietic progenitors during embryogenesis [24-26]. Adult bone marrow (BM) endothelial cells have also been shown to support the proliferation of committed myeloid and megakaryocytic progenitors in vitro [27, 28]. We previously demonstrated that coculture with a porcine brain microvascular endothelial cell line supported the expansion of adult human BM CD34 + CD38 cells as well as cells capable of providing long-term hematopoietic recovery in lethally irradiated baboons [29-31]. More recently, we showed that short-term coculture with primary human brain endothelial cells (HUBECs) caused a 4.1-fold expansion of SRCs within adult human BM [16]. Since ex vivo expansion of CB stem cells has therapeutic potential in the transplantation of adult patients, we subsequently tested whether contact or noncontact HUBEC cultures could result in a high frequency of SRCs within human CB compared with liquid suspension cultures. Using a quantitative limiting dilution analysis, we demonstrated that HUBEC coculture led to a greater SRC frequency as compared with fresh CB CD34 + cells or liquid suspension-cultured cells. Remarkably, cell-to-cell contact with HUBECs not required for SRC expansion to occur, indicating that HUBEC-secreted soluble factors accounted for this effect. These data suggest that HUBEC-based expansion protocols have the potential to improve upon current methods to expand CB stem cells for clinical transplantation. MATERIALS AND METHODS Isolation of Primary Human Brain Endothelial Cells Vessel segments (<10 cm) from the central nervous system were collected from cadavers within 12 hours postmortem under an approved tissue procurement protocol. Vessel segments were placed in complete endothelial cell culture medium containing M199 (GIBCO/BRL; Gaithersburg, MD; 10% heatinactivated fetal bovine serum (FBS; HyClone Lab; Logan, UT; µg/ml L-glutamine, 50 µg/ml heparin, 30 mg/ml endothelial cell growth supplement (Sigma; St. Louis, MO; com), 100 U/ml penicillin, and 100 µg/ml streptomycin. Vessels were incised longitudinally and oriented in such a fashion that the lumen side contacted the dish surface during in vitro culture. Well-developed endothelial cell colonies were evident by day 14 and confluent monolayers were achieved by day 30 of culture. Colonies were fed weekly with complete medium, and several passages of the primary cells were banked. Ex Vivo Culture of CB CD34 + Cells Purified human CB CD34 + cells were obtained from AllCells, LLC. (Berkeley, CA; HUBECs were subcultured at cells/well in gelatincoated 6-well plates (Costar; Cambridge, MA; corning.com) as previously described [16]. After 72 hours, HUBEC monolayers were washed with phosphate-buffered saline (PBS), and the spent medium was replaced with ex vivo expansion culture medium (5 ml/well) consisting of Iscove smodified Dulbecco s medium (IMDM; GIBCO/BRL) containing 10% FBS, 200 mm L-glutamine, 20 ng/ml TPO, 120 ng/ml SCF, and 50 ng/ml Flt-3 ligand (TSF; R&D Systems; Minneapolis, MN; in each well. Liquid suspension cultures were performed with the identical cytokines and concentrations. Purified CB CD34 + cells

3 204 Quantitative Expansion of Cord Blood Stem Cells ( ) were added to each well, and cultures were maintained at 37 C in 5% CO 2. In the HUBEC noncontact cultures, CB CD34 + cells were plated in the upper compartment of the culture well, separated from HUBEC monolayers by transwell inserts (0.4 µm; Costar). After 7-14 days of culture, nonadherent cells were harvested by washing the monolayers gently with warm complete culture medium. Immunofluorescence Staining and Analysis Human CB CD34 + cells and cultured cells were stained with monoclonal antibodies against CD34-fluorescein isothiocyanate (FITC), CD38-phycoerythrin (PE), and CXCR4- allophycocyanin (APC; Becton Dickinson; San Jose, CA; and analyzed by a fluorescence-activated cell sorter (FACSCalibur; Becton Dickinson). Controls consisted of isotype-matched monoclonal antibodies. In Vitro Methylcellulose Colony-Forming Assays Purified CB CD34 + cells and ex vivo cultured cells ( ) were cultured in 35-mm culture dishes (Miles Scientific; Naperville, IL) as previously described [16]. Culture media consisted of 1 ml IMDM, 1% methylcellulose, 30% FBS, 5 U/ml erythropoietin, 2 ng/ml GM-CSF, 10 ng/ml IL-3, and 120 ng/ml SCF. At day 14, we evaluated triplicate cultures to determine the number of colonies (>50 cells) per dish. NOD/SCID marrow cells were washed twice and placed ( ) in methylcellulose-containing culture media containing the above noted human cytokines and analyzed at day 14 for evidence of human colonies. Transplantation of Fresh CB CD34 + Cells and Ex Vivo Cultured Cells into NOD/SCID Mice NOD/SCID mice [32] were transplanted with either fresh purified CB CD34 + cells or the progeny of CB CD34 + cells that were cultured with TSF or HUBEC monolayers supplemented with TSF over a range of doses designed to achieve no engraftment in a significant fraction of mice. To avoid donor variability, CB CD34 + cell samples were pooled. For each dose range, identical donor CB CD34 + cells were used for fresh group injections as well as initiation of HUBEC and liquid suspension cultures. Cells were transplanted via tail vein injection after irradiating the NOD/SCID mice with 300 cgy using a linear accelerator source, as previously described [16]. The mice received no CD34 accessory cells or exogenous cytokines to facilitate engraftment. Mice were sacrificed at week 8, and marrow samples were obtained by flushing their femurs and tibias with IMDM at 4 C. Flow cytometric analysis of NOD/SCID marrow cells was performed at week 8, as previously described, using commercially available monoclonal antibodies against human leukocyte differentiation antigens to identify engrafted human leukocytes and discriminate their hematopoietic lineages [33]. Immunofluorescence staining of marrow cells was performed following our previously published procedures [16]. Statistical Analysis For purposes of our limiting dilution assays, we scored a transplanted mouse as positively engrafted if 1% of the marrow cells expressed human CD45 via FACS analysis. This criterion is consistent with previously published limiting dilution analyses of human cell repopulation of NOD/SCID mice [20, 34]. SRC frequency in each cell source was calculated using the maximum likelihood estimator, as described previously by Taswell [35] for the single-hit Poisson model [17, 35, 36]. The χ 2 variable provides a measure of the legitimacy of using pooled data and of the validity of applying the single-hit model [17, 36]. We calculated confidence intervals for the frequencies using the profile likelihood method, and we used the likelihood ratio test to confirm the fit of the model. As a confirmation of the maximum likelihood estimator, we also applied a minimum χ 2 estimator to the pooled data. Assessment of the Seeding Efficiency of Fresh CB CD34 + Cells Versus Ex Vivo Cultured CB Cells Analysis of the seeding efficiency of human CB CD34 + cells to the marrow of NOD/SCID mice was performed following the method of van Hennik et al. [34]. Briefly, NOD/SCID mice were irradiated with 350 cgy of total body irradiation and then were injected with either fresh CB CD34 + cells, the progeny of CB CD34 + cells cultured with TSF for 7 days, or the progeny of CB CD34 + cells cultured with HUBECs + TSF for 7 days. Eight mice were injected in each group. Mice were sacrificed at 24 hours postinjection, bilateral femurs were flushed, and marrow cells were collected. Marrow red blood cells were depleted by 10- minute incubation with RBC lysis buffer (Sigma), and postlysis cell counts were obtained. The cells were then resuspended in PBS supplemented with 10% FBS, 2% normal human serum, and 1% penicillin/streptavidin, and stained with anti-human CD45-FITC and anti-human CD34-PE (Becton Dickinson). 7-AAD (Becton Dickinson) was used to exclude dead cells. Appropriate isotype control monoclonal antibodies were used for each fluorochrome. Cells within the NOD/SCID marrow that expressed both human CD45 and human CD34 were considered positively seeded at 24 hours. For the purposes of calculating the seeding efficiency of human CD34 + cells to the marrow, we estimated that each femur contained 6% of the total BM cellularity, as previously described [34]. The number of retrieved CD34 + cells was divided by the number of infused CD34 + cells,

4 Chute, Muramoto, Fung et al. 205 corrected for the purity of the population. Seeding efficiency was expressed as a percentage. For the purposes of the seeding efficiency analyses, a minimum of 750,000 flow cytometry events was analyzed per each sample. RESULTS HUBEC Coculture Supported the Expansion of CB CD34 + Cells and Colony-Forming Cells Primary HUBECs demonstrated a cobblestone morphology in culture, and >90% expressed human von Willebrand factor, as previously described [16]. The effects of liquid suspension culture, HUBEC coculture, and noncontact HUBEC culture on the in vitro expansion of CB CD34 + cells and colony-forming cells (CFCs) were compared. All cultures were supplemented with TSF, as noted in Materials and Methods. HUBEC coculture supported a 26-fold increase in total cells, a 19-fold increase in CD34 + cells, and a 156-fold increase in CD34 + CD38 cells after 7 days (Table 1). As shown in a representative experiment (Fig. 1), CB CD34 + CD38 cells increased from a mean 11.2% of the day-0 CB CD34 + population to 66.9% of the total population at day 7 (Fig. 1A, 1B). Similarly, HUBEC noncontact cultures supported a 32-fold increase in total cells, a 14-fold increase in CD34 + cells, and a 115-fold increase in CD34 + CD38 cells (Fig. 1C). In contrast, liquid suspension cultures supplemented with TSF supported a 16-fold increase in total cells, but we observed a decline in the percentage of CD34 + and CD34 + CD38 cells, resulting in a 5.4-fold increase in CD34 + cells and a 10-fold increase in CD34 + CD38 cells by day 7 (Fig. 1D). HUBEC coculture induced a 73-fold increase in colonyforming units-granulocyte-macrophage (CFU-GM), an 11- fold increase in BFU-E, and a 23-fold increase in CFU-mix compared with day-0 CB CD34 + cells (Table 1). HUBEC noncontact cultures supported a comparable expansion of total CFCs, with a greater proportion of BFU-E than contact HUBEC cultures. Liquid suspension cultures also induced expansion of CFCs, but the expansion of CFU-GM and CFU-total was less than 50% of the expansion observed following HUBEC coculture. HUBEC Coculture Increased the SCID-Repopulating Capacity of CB CD34 + Cells NOD/SCID mice were transplanted with either fresh CB CD34 + cells (n = 37 mice), CB CD34 + cells cultured with TSF (n = 45 mice), or HUBEC-cultured cells (n = 46 mice) over a range of doses designed to achieve nonengraftment in a fraction of mice. As shown in Figure 2A, transplantation of fresh CB CD34 + cells resulted in no engraftment in eight mice. Similarly, the progeny of CB CD34 + cells cultured with TSF also failed to engraft in 10 mice. The progeny of CB CD34 + cells cultured with HUBECs engrafted in 1 of 10 mice (10%) (Fig. 2A). Over a dose range of to cells, fresh CB CD34 + cells engrafted in only 4 of 16 mice (25%), whereas the progeny of to CB CD34 + cells cultured with TSF engrafted in 9 of 19 mice (47%). In contrast, over the same dose range, the progeny of HUBEC-cultured CB CD34 + cells engrafted in 14 of 20 mice (70%) (Fig. 2B, 2C). Similarly, at a dose of , the progeny of HUBEC cultures showed twofold higher human CD45 + cell engraftment than either fresh CB CD34 + cells or TSF-cultured cells at the identical dose. At a dose of CB CD34 + cells, 100% of mice transplanted with either fresh CB CD34 + cells or HUBEC-cultured cells showed engraftment, indicating that nonlimiting numbers of SRCs were present in both groups at this dose. The progeny of CB CD34 + cells cultured with TSF engrafted in 7 of 8 mice (88%) (Fig. 2D). Table 1. HUBEC coculture supports greater expansion of human CB progenitor cells n cells procured 10 5 n CFCs 10 3 ConditionsCell yield CD34 + CD34 + CD38 CFU-GM BFU-E CFU-Mix CFU total Day ± ± ± ± ± ± 2.2 HUBEC 26.0 ± ± ± ± ± ± ± 24.0 (26-fold) (19-fold) (156-fold) HUBEC 32.2 ± ± ± ± ± ± ± 25.4 (noncontact) (32-fold) (14-fold) (115-fold) TSF alone 16.0 ± ± ± ± ± ± ± 25.0 (16-fold) (5.4-fold) (10-fold) Purified human CB CD34 + cells ( ) were plated (n = 5), as described in Materials and Methods. Cells were harvested on day 7 of culture and analyzed via flow cytometry. Numbers in parentheses in the cell yield, CD34 +, and CD34 + CD38 columns indicate the fold increases in these populations following culture. CFC values reflect the total number of CFCs per culture condition. Values are means ± standard deviations of five cultures.

5 206 Quantitative Expansion of Cord Blood Stem Cells Figure 1. Phenotypic analysis of CB CD34 + cells at day 0 and following ex vivo culture. Purified human CB CD34 + cells were plated on confluent HUBEC monolayers, noncontact HUBEC cultures, and liquid suspension cultures in the presence of optimal concentrations of TPO + SCF + Flt-3 ligand. A) The phenotype of untreated CB CD34 + cells at day 0. B) HUBEC-cultured CB cells at day 7, demonstrating a high percentage of CD34 + CD38 cells. C) Noncontact HUBEC-cultured CB cells at day 7, showing preservation of the CD34 + CD38 phenotype. D) Liquid suspension cultures, demonstrating a decline in CD34 + and CD34 + CD38 cells at day 7. Log fluorescence distribution of CD34 expression is shown along the x-axis, and CD38 expression along the y-axis. Isotype matched control monoclonal antibody staining is shown at the left of each figure. The numbers within these quadrants indicate the percentage of total cells that fall within the particular quadrant. A B C IgG PE IgG PE In order to assess whether cellto-cell contact was necessary for the expansion of SRCs during HUBEC culture, we also transplanted NOD/ SCID mice with the progeny of CB CD34 + cells that were cultured with HUBECs in the absence of cell-tocell contact. As shown in Figure 2C, the progeny of CB CD34 + cells plated in noncontact HUBEC cultures repopulated 8 of 9 mice (89%) and at levels of human D CD45 + cell engraftment (mean, 6.4% HuCD45 + ) that were comparable with or higher than those observed with the progeny of contact HUBEC-cultured cells transplanted at the same dose. These data confirmed that cell-to-cell contact was not required for HUBECs to sustain SRCs in culture. In order to determine whether HUBEC coculture could maintain or expand SRCs beyond 7 days, we also transplanted NOD/SCID mice with the progeny of CB CD34 + cells following 14 days of HUBEC coculture. As shown in Figure 2C, the progeny of day-14 HUBEC cultures repopulated 100% of mice with a 4.2-fold higher IgG PE IgG PE IgG FITC IgG FITC CD38 PE CD38 PE CD38 PE IgG FITC CD38 PE IgG FITC CD34 FITC CD34 FITC CD34 FITC CD34 FITC HuCD45 + cell engraftment (mean, 14.0% HuCD45 + cells) than in mice transplanted with day-7 HUBEC-cultured cells. These results indicate that enrichment of CB SRCs continued to occur during HUBEC coculture through day

6 Chute, Muramoto, Fung et al. 207 A % Human cell engraftment C % Human cell engraftment Fresh TSF HUBECcultured CB CD34 + B % Human cell engraftment D % Human cell engraftment Fresh TSF HUBECcultured CB CD Fresh TSF HUBECcultured HUBEC HUBEC CB CD34 + Transwell day 14 Fresh TSF HUBECcultured CB CD34 + Figure 2. Human cell repopulation in NOD/SCID mice transplanted with limiting doses of CB CD34 + cells and their cultured progeny. The level of human cell engraftment at 8 weeks was determined by flow cytometric analysis of human CD45 expression on cells within the NOD/SCID marrow. A) CB CD34 + cells (left) or their progeny following TSF culture (middle) or HUBEC coculture (right) 7 days were transplanted into NOD/SCID mice. Percent human CD45 + cell engraftment is shown on the y-axis. B) Human cell engraftment following transplantation of CB CD34 + cells or their progeny. C) Human cell engraftment following transplantation of CB CD34 + cells or their progeny. At the second from right, human cell engraftment is shown in mice transplanted with the progeny of CB CD34 + cells cultured for 7 days with HUBEC under noncontact conditions. At the far right, human cell engraftment is shown in mice transplanted with the progeny of CB CD34 + cells following 14 day coculture with HUBECs. D) Human cell engraftment following transplantation of CB CD34 + cells or their progeny. HUBEC-Cultured CB Cells Demonstrated Multilineage Differentiation In Vivo Figure 3 shows human CD45 + cell engraftment at week 8 in representative mice that were transplanted with fresh CB CD34 + cells ( ) (Fig. 3A), the progeny of the same dose of CB CD34 + cells cultured with TSF (Fig. 3B), and the same dose of CB CD34 + cells cultured with HUBEC monolayers (Fig. 3C). As shown in Figure 3D, mice that were transplanted with limiting doses of HUBEC-cultured cells displayed myeloid and B-lymphoid differentiation, indicating that highly primitive repopulating cells were maintained during HUBEC coculture. The proportions of human CD45 + cells expressing CD34, CD19, and CD13 were comparable in mice that had engrafted with fresh CB CD34 + cells, HUBEC-cultured cells, and TSF-cultured cells (Table 2). Both TSF-cultured and HUBEC-cultured cells produced modestly higher levels of CD13 + myeloid differentiation in vivo compared with fresh CB CD34 + cell grafts, but this difference was not statistically significant. HUBEC Coculture Increased SRC Frequency Within Human CB CD34 + Cells In order to estimate the frequency of SRCs within the three CB populations studied, we pooled data from the limiting dilution assays of fresh CB CD34 + cells, TSF-cultured cells, and HUBEC-cultured cells, according to methods previously described [16, 35, 36]. We calculated the frequency of SRCs using the maximum likelihood estimator [36]. The value of χ 2 in all cases was not statistically significant (p > 0.6), verifying the internal consistency of our assays and allowing pooling of the data. The frequency of SRCs within fresh CB CD34 + cells was 1 in 21,000 cells (95% confidence interval [CI], 1/11,000-1/43,000) (Fig. 4A). The SRC frequency within cells

7 208 Quantitative Expansion of Cord Blood Stem Cells A IgG PerCP B IgG PerCP C IgG PerCP Mu IgG FITC Mu IgG FITC Mu IgG FITC 0.0 Mu CD45 FITC 0.1 Mu CD45 FITC 2.8 Mu CD45 FITC D IgG PerCP I Mu IgG FITC III IgG PE v Hu CD19 PE 11.0 II Mu CD13 FITC IV Hu CD34 PE vi Mu CD13 FITC Figure 3. Phenotypic analysis of engraftment and multilineage differentiation of HUBEC-cultured CB cells in NOD/SCID bone marrow. A) Expression of human CD45 + cells within the BM of a representative NOD/SCID mouse transplanted with CB CD34 + cells. B) Expression of human CD45 + cells in a mouse transplanted with the progeny of CB CD34 + cells following culture with TSF alone. C) Expression of human CD45 + cells in a mouse transplanted with the progeny of CB CD34 + cells following HUBEC coculture. Percent human CD45 + cell engraftment is shown in upper left quadrants. Isotype controls are shown at left. D) Lineage distribution of engrafted human cells within a representative mouse that was transplanted with HUBEC-cultured CB cells. (D-I) Staining with isotype control PerCP and FITC monoclonal antibodies. (D-II) Panel of HuCD45 and murine-cd45 staining, demonstrating presence of human and murine cells. (D-III) Isotype control PerCP versus PE staining. (D-IV) Dual expression of human CD45 and human CD34 on engrafted cells within the murine marrow. (D-V) Dual expression of CD45 and CD19 on engrafted human cells. (D-VI) Dual expression of CD45 and CD13 on engrafted human cells. Percentages of human cells of each lineage are shown within each quadrant. Table 2. HUBEC-cultured cells generate multilineage human cell differentiation in vivo Percentage of engrafted CD45 + cells expressing: Culture condition CD34 CD19 CD13 Fresh CB CD ± ± ± 11.2 TSF-cultured 20.8 ± ± ± 11.6 HUBEC-cultured 18.1 ± ± ± 11.8 NOD/SCID mice were transplanted with to fresh CB CD34 + cells or the progeny of these cells following 7 days of HUBEC coculture or TSF alone, as described in Materials and Methods. The numbers shown under the CD34, CD19, and CD13 columns indicate the percentage of engrafted HuCD45 + cells that expressed the particular lineage antigen. Only mice that demonstrated >1% HuCD45 + cell engraftment were analyzed for multilineage differentiation. Values are means ± standard deviations of percentage engraftment in 10 mice per group. cultured with TSF alone was 1 in 16,000 (CI, 1/9,700-1/29,000; Fig. 4B). The SRC frequency within HUBECcultured cells was significantly higher, at 1 in 6,100 (CI, 1/3,700-1/11,000; Fig. 4C). Therefore, HUBEC coculture supported a 3.4-fold greater number of SRCs than fresh CB CD34 + cells and 2.6-fold more SRCs than TSF-cultured cells. Applying the likelihood ratio test, the SRC frequency calculated within HUBEC-cultured cells was significantly higher than the SRC frequency in either fresh CB CD34 + cells or TSF-cultured cells (p = and p = 0.013, respectively). As further confirmation of the validity of applying the single-hit Poisson model to our limiting dilution assay, we also estimated the frequency of SRCs using the minimum χ 2 estimation [36]. Again, the χ 2 value was not significant in all cases (p > 0.6).

8 Chute, Muramoto, Fung et al. 209 A Fraction of negative mice B Fraction of negative mice C Fraction of negative mice , , , , ,000 n of transplanted CB CD34 + cells , , , , ,000 n of transplanted cells (initial CD34 + cell equivalent) , , , , ,000 n of transplanted cells (initial CD34 + cell equivalent) Figure 4. HUBEC coculture increased the frequency of SRC within human cord blood. A) NOD/SCID mice (n = 37) were transplanted with fresh CB CD34 + cells over a range of doses, and the engraftment frequencies at each dose were plotted. The resultant curve indicates the estimated frequency of SRCs within this population. B) NOD/SCID mice (n = 45) were transplanted with the progeny of CB CD34 + cells following culture with TPO + SCF + Flt-3 ligand. The resultant curve indicates the frequency of SRCs within this population. C) NOD/SCID mice (n = 46) were transplanted with the progeny of CB CD34 + cells following culture with HUBEC monolayers supplemented with TPO + SCF + Flt-3 ligand. The resultant curve indicates the frequency of SRCs within the HUBEC-cultured population. The numbers shown within each box indicate the calculated frequency of SRCs using the maximum likelihood estimator. HUBEC Coculture Increased the Engraftment of Human CB CD34 + Cells in the NOD/SCID Marrow In order to determine whether the greater SCID-repopulating capacity of HUBEC-cultured CB cells was a function of improved seeding in the recipient BM following transplantation, we performed hematopoietic cell seeding efficiency studies, as previously described by van Hennik et al. [34]. Figure 5A shows a representative flow cytometric analysis utilized to determine the percentage of human CD34 + cells engrafted within the NOD/SCID marrow compartment 24 hours posttransplantation. Representative engraftment of human CD34 + cells in the NOD/SCID marrow 24 hours following tail vein injection of fresh CB CD34 + cells is shown. Nonviable cells were excluded using 7-AAD staining, and CD34 PE-positive cells with low side scatter were gated (Fig. 5A). These cells were then analyzed for CD45 expression as well as high forward scatter. Figure 5A shows that a population of human cells (0.02% of total gated population) that stained positively for both HuCD34 and HuCD45 was evident within the mouse BM following transplantation with fresh CB CD34 + cells. As shown in Figure 5B, culture with TSF alone for 7 days resulted in greater seeding of CD34 + cells within the marrow at 24 hours compared with fresh CB CD34 + cells. Mice transplanted with HUBEC-cultured cells showed the highest levels of CD34 + cell engraftment at 24 hours, as shown in Figure 5C. Applying the method described by van Hennik et al. [34], we calculated that mice transplanted with HUBEC-cultured cells had 7.5-fold and 4.0-fold higher numbers of engrafted CD34 + cells in the marrow than did those transplanted with fresh CB CD34 + cells and TSF-cultured cells, respectively. As shown in Figure 6A, the differences in the engraftment of HUBEC-cultured CB CD34 + cells versus fresh CB CD34 + cells and versus TSFcultured CD34 + cells were highly significant (p = 0.003, p = 0.003, respectively; Wilcoxon rank sum test) and correlated with the SRC frequency estimates for each group. However, when we corrected for the larger numbers of CD34 + cells infused within the HUBEC-culture group and the TSF-culture group, the seeding efficiencies (n of CD34 + cells engrafted at 24 hours/n of CD34 + cells infused) of HUBECcultured grafts and TSF-cultured grafts were both lower than the seeding efficiency of fresh CB CD34 + cells (2.1% versus 1.8% versus 3.9%, respectively; Fig. 6B), although these differences were not statistically significant (p > 0.2 for both; Wilcoxon rank sum test). Since the homing of human hematopoietic stem cells to the marrow compartment has been previously associated with the surface expression of the chemokine receptor CXCR4 on human CD34 + cells [37], we also measured the CXCR4 surface expression levels on fresh CB CD34 + cells

9 210 Quantitative Expansion of Cord Blood Stem Cells A B C HuCD34 PE HuCD34 PE HuCD34 PE ,000 Side scatter ,000 Side scatter ,000 Side scatter HuCD45 FITC HuCD45 FITC HuCD45 FITC ,000 Forward scatter ,000 Forward scatter ,000 Forward scatter HuCD34 PE HuCD34 PE HuCD34 PE 0.02% HuCD45 FITC 0.05% HuCD45 FITC 0.2% HuCD45 FITC Figure 5. Flow cytometric analysis demonstrating the seeding efficiencies of fresh CB CD34 + cells, TSF-cultured cells, and HUBEC-cultured cells to the NOD/SCID marrow. A) Representative seeding of human CD34 + cells in the NOD/SCID marrow 24 hours following infusion of fresh CB CD34 + cells. Dead cells were excluded first using 7-AAD. In the dot plot at left, viable CD34-PE + cells with low side scatter were gated. These cells were then gated for CD45-FITC positivity and large forward scatter properties (middle). At right, the double-positive CD34 + CD45 + population of CB cells is shown, demonstrating a definitive population of human cells engrafted in the NOD/SCID marrow at 24 hours. B) Representative seeding of human CD34 + cells in the NOD/SCID marrow following infusion of the progeny of CB CD34 + cells cultured with TSF 7 days. A higher frequency of engrafted CD34 + CD45 + cells is shown. C) Representative seeding of human CD34 + cells in the NOD/SCID marrow following transplantation of the progeny of CB CD34 + cells cocultured with HUBEC 7 days. A significantly higher percentage of engrafted CD34 + CD45 + cells is shown. versus HUBEC-cultured cells versus TSF-cultured cells. Fresh CB CD34 + cells had a relatively low level of surface expression of CXCR4 (mean, 16.9%), whereas TSF culture resulted in a 4.2-fold greater CXCR4 expression on CD34 + cells. HUBEC coculture resulted in a 2.9-fold greater CXCR4 expression. Both TSF-cultured and HUBEC-cultured CD34 + cells showed significantly higher surface expression levels of CXCR4 than fresh CB CD34 + cells (p = and p = 0.02, respectively; Wilcoxon rank sum test). Figure 7 shows the FACS analysis of CXCR4 on fresh CB CD34 + cells, TSF-cultured cells, and HUBEC-cultured cells from a representative experiment. Although TSF-cultured CD34 + cells and HUBEC-cultured CD34 + cells had significantly higher surface expression levels of CXCR4, these grafts surprisingly showed lower seeding efficiencies of CD34 + cells to the NOD/SCID marrow compared with fresh CB CD34 + cells. DISCUSSION Umbilical cord blood has been successfully utilized as a source of transplantable stem cells in the treatment of children and adolescents with both malignant and nonmalignant diseases [2, 4, 7, 38]. Cord blood could also be an advantageous alternative source of stem cells for the treatment of adults who meet the indications for stem cell transplantation, but the limited stem cell numbers per CB graft

10 Chute, Muramoto, Fung et al. 211 A n of engrafted CD34 + cells ( 10 3 ) B % Seeding efficiency Fresh TSF HUBECcultured CB CD34 + Fresh TSF HUBECcultured CB CD34 + Figure 6. Total engraftment and seeding efficiency of human CB CD34 + cells and cultured CB cells in the NOD/SCID marrow. A) The mean numbers of engrafted CD34 + cells following infusion of fresh CB CD34 + cells versus the progeny of CB CD34 + cells cultured with TSF 7 days versus the progeny of 7-day HUBEC coculture are shown (n = 8 per group). B) The mean seeding efficiencies (n of infused CD34 + cells/n of retrieved CD34 + cells) of fresh CB CD34 + cells, TSF-cultured cells, and HUBEC-cultured cells are shown. * indicates a significant difference between HUBEC-cultured cells and fresh CB CD34 + cells or TSF-cultured cells. have hindered its use [4, 6-8]. Over the past decade, several preclinical studies have suggested that CB CD34 + cells, unlike adult BM stem cells, could be expanded in vitro under various conditions while maintaining SRCs [17-20]. Bhatia et al. demonstrated that CB SRCs could be expanded two- to fourfold by day 4 of culture with SCF + Flt-3 ligand + IL-3 + IL-6 + G-CSF, but all SRCs were lost by day 9 [17]. Conneally et al. reported a comparable twofold expansion of CB-competitive repopulating units following 5-8 days of culture with the identical cytokine combination [18]. Subsequently, the combination of SCF + Flt-3 ligand + MGDF + IL-6 was found to expand CB SRCs for up to 10 weeks in stroma-free cultures [19]. A more recent study by Tanavde et al. [21] confirmed the maintenance of CB SRCs over 4 weeks of culture with SCF + Flt-3 ligand + TPO, but these authors did not observe a quantitative expansion of SRCs using this combination [21]. These preclinical studies raised optimism regarding the * potential application of ex vivo expanded CB grafts in the transplantation of patients. However, two recent clinical trials indicated that CB grafts cultured under stroma-free conditions did not shorten the time to hematopoietic recovery in transplanted patients [13, 23]. Therefore, additional strategies to expand CB stem and progenitor populations are warranted. In this study, we examined whether coculture with primary HUBECs could support a higher frequency of SRCs than fresh CB CD34 + cells or cells optimally cultured with TSFalone. HUBEC coculture plus TSF resulted in a 3.4-fold greater SRC frequency by limiting dilution analysis, whereas liquid suspension cultures supplemented with TSF alone supported only a 1.3-fold greater SRC frequency than fresh CB CD34 + cells. Transplantation of NOD/SCID mice with day-14 HUBECcultured cells resulted in 4.2-fold higher levels of HuCD45 + cell engraftment compared with day-7 HUBEC-cultured cells, suggesting that repopulating cell self-renewal continued to occur through day 14. These results demonstrate that primary HuBECs provide a hematopoietic activity that uniquely promotes human CB expansion and the self-renewal of long-term repopulating cells. Previous studies have indicated that contact between hematopoietic stem cells and stromal cells or fibronectin can prevent the loss of stem cell quality that occurs during ex vivo exposure to cytokines [39, 40]. Recently, cell-to-cell contact was shown to be essential for the maintenance of human myeloid-lymphoid-initiating cells in coculture with a murine stromal cell line, AFT024 [41]. Similarly, a murine stromal cell line, HESS-5 [42], and a human BM stromal cell line infected with the human telomerase catalytic subunit (htert) gene have been shown to support the maintenance of CB SRCs via contact-mediated coculture [43]. In contrast to these reports, we observed that noncontact HUBEC cultures expanded CD34 + cells, CFCs, and most importantly, SRCs at levels comparable with those observed within contact HUBEC cultures. Although we did not perform a limiting dilution analysis to quantify the SRC expansion within noncontact HUBEC cultures, our data indicate that exposure of CB CD34 + cells to HUBEC-conditioned media significantly improved their repopulating capacity over cells cultured with TSF alone. These data demonstrate that HUBEC, unlike other described cell lines, elaborate a soluble hematopoietic activity that maintains proliferating stem cells independently of cell-to-cell contact. The application of a HUBEC-conditioned media in the expansion of CB grafts would be clinically advantageous, since the concern regarding contamination with endothelial cells in patient CB grafts would be eliminated. We are currently performing subtractive protein fractionation studies on HUBEC-conditioned media in order to isolate the soluble

11 212 Quantitative Expansion of Cord Blood Stem Cells Figure 7. CXCR4 surface expression on CB CD34 + cells. Representative histograms are shown demonstrating the surface expression of CXCR4 and CD34 on day-0 CB CD34 + cells (A), day-7 TSF-cultured cells (B), and day-7 HUBEC-cultured cells (C) (n = 5). Isotype controls for each sample are shown at the left of each histogram. The percentages inside the charts indicate the percentage of total cells that fall within the particular quadrant. A IgG APC 10 4 R14 R R16 R17 CXCR4 APC 10 R14 R R16 R17 7% 91% proteins responsible for the hematopoietic activity we have observed. Unrelated CB transplantation has been associated with slower platelet and neutrophil recovery compared with unrelated BM transplants [2-4]. Guenechea et al. [44] experimentally demonstrated that ex vivo expansion of CB CD34 + cells under stroma-free conditions resulted in significant delays in human CD45 + cell repopulation in NOD/SCID mice compared with fresh CB CD34 + cells. The preclinical results reported by Guenechea et al. predicted, in part, the results of two recent clinical trials that showed that CB CD34 + cells expanded in liquid suspension cultures failed to shorten platelet or neutrophil recovery compared with historical controls that utilized fresh CB grafts [13, 23]. The results of our seeding efficiency analysis indicate that B IgG APC C IgG APC transplantation of HUBEC-cultured CB cells significantly improves the immediate engraftment of human CD34 + cells in the NOD/SCID marrow compared with TSF-cultured or fresh CB CD34 + cells. It is plausible that the engraftment of greater numbers of HUBEC-cultured CB CD34 + cells could translate into earlier hematopoietic recovery in the clinical setting when compared with fresh CB CD34 + cell grafts or liquid suspension-cultured cells. HUBEC-cultured CB grafts appear to provide both earlier CD34 + cell engraftment and improved long-term repopulation as measured in the NOD/SCID model. However, since we did not formally measure the kinetics of HUBEC-cultured cell repopulation in this study, we cannot conclude that HUBEC-cultured CB R14 R R16 R17 IgG FITC R14 R R16 IgG FITC R IgG FITC CXCR4 APC CXCR4 APC R14 R CD34 FITC cells would provide earlier hematopoietic recovery. We are planning to test the efficacy of HUBEC-cultured CB cells in providing earlier hematopoietic recovery and long-term repopulation in a phase I clinical trial. The seeding efficiency of normal human CB cells to the NOD/SCID marrow compartment has been shown to be low, with only 1.4%-2.8% of transplanted CD34 + cells detectable in the marrow 24 hours postinfusion [34]. Augmentation of seeding or homing mechanisms, particularly via upregulation of CXCR4 on CB CD34 + CD38 cells, has been associated with improved long-term repopulation in the NOD/SCID model [45, 46]. In this study, we performed seeding efficiency studies in order to determine whether differences in R16 R R14 R15 R16 15% CD34 FITC R17 5% 10 0 CD34 FITC 35% 22%

12 Chute, Muramoto, Fung et al. 213 the immediate engraftment capacities of fresh CB CD34 + cells versus TSF-cultured cells versus HUBEC-cultured cells contributed to the differences we observed in SRC frequencies. Our results indicate that the number of CD34 + cells engrafted at 24 hours following transplantation of HUBECcultured cells was significantly higher than the engraftment numbers observed in mice transplanted with TSF-cultured grafts or fresh CB CD34 + cells. The higher level of seeded CD34 + cells within the HUBEC-culture group correlated qualitatively with the calculated higher SRC frequency within this group compared with both TSF-cultured cells and fresh CB CD34 + cells. However, when corrected for the total number of CD34 + cells infused, we found that fresh CB CD34 + cells had a modestly higher seeding efficiency than HUBECcultured cells and TSF-cultured cells. These seeding efficiencies were surprising in light of the significantly higher surface expression levels of CXCR4 on TSF-cultured and HUBEC-cultured CD34 + cells compared with fresh CB CD34 + cells (70.2% versus 49.4% versus 16.9%, respectively). Previous studies have indicated that CB CD34 + cells home to the marrow in a CXCR4-dependent manner [37, 45], but our results suggest, as have other reports [46, 47], that CXCR4 surface expression is not obligatory for human hematopoietic cell repopulation in NOD/SCID mice. Our analysis indicates that the greater repopulating capacity of HUBEC-cultured cells cannot be accounted for on the basis of better homing mechanisms. Our data alternatively suggest that HUBECcultured grafts would necessarily have to contain a higher number of long-term repopulating cells than fresh CB CD34 + cell grafts or TSF-cultured grafts to account for the higher SRC frequency that we observed. We have not yet delineated the specific mechanisms through which HuBECs affect the ex vivo expansion of hematopoietic repopulating cells. Several recent studies have suggested important roles for candidate proteins in the maintenance of hematopoietic stem cells in vitro, including Notch ligands [48], stromal cell-derived factor 1 [49], sonic hedgehog [50], and most recently, the Wnt3a protein [51]. In addition, the contribution of endothelial-cell-derived proteins toward hematopoietic stem cell proliferation has been suggested by the demonstration that murine embryonic aortagonad-mesonephros region-derived endothelial cells could support the in vitro maintenance of murine stem cells [52]. More recently, Terskikh et al. [53] reported that the genetic programs within hematopoietic stem cells and neural stem cells are overlapping. Those results suggest a significant probability that brain endothelial cells may elaborate growth factors important to hematopoietic stem cells. We are currently performing subtractive gene expression analyses on HUBECs versus nonhematopoietically active endothelial cell lines to identify candidate gene products that are differentially expressed by the brain endothelial cells. Since HUBECs are primary, homogeneous endothelial cells, as opposed to heterogeneous stromal cells, we anticipate that such an analysis, in combination with protein fractionation of HUBEC-conditioned media, will identify unique factors that are critically involved in stem cell maintenance and self-renewal. Although recent progress in the experimental expansion of human CB CD34 + cells and SRCs has raised optimism for broadened application of CB transplantation in the treatment of adults, this progress has not yet translated into meaningful improvements in time to engraftment or immune reconstitution in patients [13, 23]. This study indicates that the application and further development of HUBEC coculture or a HUBEC-derived conditioned medium can potentially improve the engraftment and long-term repopulation of transplanted human CB stem cells in vivo. The successful translation of this culture methodology to clinical expansion protocols may allow the utilization of cord blood grafts in the treatment of many adult patients who currently are not considered candidates. ACKNOWLEDGMENTS We thank Dr. David Venzon for his critical assistance with the statistical analysis. The HUBEC cells were kindly provided by the Naval Medical Research Center (NMRC), Silver Spring, MD, as per the Cooperative Research and Development Agreement #NCRADA-NMRDC/NMRI/ Biosource between NMRC and Large Scale Biology Corporation. REFERENCES 1 Broxmeyer HE, Gluckman E, Auerbach A et al. Human umbilical cord blood: a clinically useful source of transplantable hematopoietic stem/progenitor cells. Int J Cell Cloning 1990;8(suppl 1): Rocha V, Cornish J, Sievers E et al. Comparison of outcomes of unrelated bone marrow and umbilical cord blood transplants in children with acute leukemia. Blood 2001;97: Rocha V, Wagner J, Sobocinski K et al. Graft-versus-host disease in children who have received a cord-blood or bone marrow transplant from an HLA-identical sibling. Eurocord and International Bone Marrow Transplant Registry Working Committee on Alternative Donor and Stem Cell Sources. N Engl J Med 2000;342: Cairo MS, Wagner J. Placental and/or umbilical cord blood: an alternative source of hematopoietic stem cells for transplantation. Blood 1997;90:

13 214 Quantitative Expansion of Cord Blood Stem Cells 5 Broxmeyer H, Douglas G, Hangoc C et al. Human umbilical cord blood as a potential source of transplantable hematopoietic stem/progenitor cells. Proc Natl Acad Sci USA 1989;86: Wagner JE, Rosenthal J, Sweetman R et al. Successful transplantation of HLA-matched and HLA-mismatched umbilical cord blood from unrelated donors: analysis of engraftment and acute graft-versus-host-disease. Blood 1996;88: Kurtzberg J, Laughlin M, Graham ML et al. Placental blood as a source of hematopoietic stem cells for transplantation into unrelated recipients. N Engl J Med 1996;335: Balduzzi A, Gooley T, Anasetti C et al. Unrelated donor marrow transplantation in children. Blood 1995;86: Locatelli F, Rocha V, Reed W et al. Related umbilical cord blood transplantation in patients with thalassemia and sickle cell disease. Blood 2003;101: Wagner J, Kernan N, Steinbuch M et al. Allogeneic sibling umbilical-cord-blood transplantation in children with malignant and non-malignant disease. Lancet 1995;346: Gluckman E, Rocha V, Boyer-Chammard A et al. Outcome of cord-blood transplantation from related and unrelated donors. Eurocord Transplant Group and the European Blood and Marrow Transplantation Group. N Engl J Med 1997;337: McNiece I, Briddell R. Ex vivo expansion of hematopoietic progenitor cells and mature cells. Exp Hematol 2001;29: Jaroscak J, Goltry K, Smith A et al. Augmentation of umbilical cord blood (UCB) transplantation with ex vivo expanded UCB cells: results of a phase I trial using the AastromReplicell System. Blood 2003;101: Bhatia M, Wang J, Kapp U et al. Purification of primitive human hematopoietic cells capable of repopulating immunedeficient mice. Proc Natl Acad Sci USA 1997;94: Dorrell C, Gan O, Pereira D et al. Expansion of human cord blood CD34 + CD38 cells in ex vivo culture during retroviral transduction without a corresponding increase in SCID repopulating cell (SRC) frequency: dissociation of SRC phenotype and function. Blood 2000;95: Chute JP, Saini A, Chute D et al. Ex vivo culture with human brain endothelial cells increases the SCID repopulating capacity of adult human bone marrow. Blood 2002;100: Bhatia M, Bonnet D, Kapp U et al. Quantitative analysis reveals expansion of human hematopoietic-repopulating cells after short-term ex vivo culture. J Exp Med 1997;186: Conneally E, Cashman J, Petzer A et al. Expansion in vitro of transplantable human cord blood stem cells demonstrated using a quantitative assay of their lymphoid-myeloid repopulating activity in nonobese diabetic-scid/scid mice. Proc Natl Acad Sci USA 1997;94: Piacibello W, Sanavio F, Severino A et al. Engraftment in nonobese diabetic severe combined immunodeficient mice of human CD34 + cord blood cells after ex vivo expansion: evidence for the amplification and self-renewal of repopulating stem cells. Blood 1999;93: Ueda T, Tsuji K, Yoshino H et al. Expansion of human NOD/SCID-repopulating cells by stem cell factor, Flk2/Flt3 ligand, thrombopoietin, IL-6, and soluble IL-6 receptor. J Clin Invest 2000;105: Tanavde V, Malehorn M, Lumkul R et al. Human stem-progenitor cells from neonatal cord blood have greater hematopoietic expansion capacity than those from mobilized adult blood. Exp Hematol 2002;30: McNiece I, Almeida-Porada G, Schpall E et al. Ex vivo expanded cord blood cells provide rapid engraftment in fetal sheep but lack long-term engrafting potential. Exp Hematol 2002;30: Shpall E, Quinones R, Giller R et al. Transplantation of ex vivo expanded cord blood. Biol Blood Marrow Transplant 2002;8: Shalaby F, Rossant J, Yamaguchi TP et al. Failure of blood island formation and vasculogenesis in flk-1 deficient mice. Nature 1995;376: Choi K, Kennedy M, Kazarov A et al. A common precursor for hematopoietic and endothelial cells. Development 1998;125: Oberlin E, Tavian M, Blazsek I et al. Blood-forming potential of vascular endothelium in the human embryo. Development 2002;129: Rafii S, Shapiro F, Rimarachin J et al. Isolation and characterization of human bone marrow microvascular endothelial cells: hematopoietic progenitor cell adhesion. Blood 1994;84: Raffi S, Shapiro F, Pettengell R et al. Human bone marrow microvascular endothelial cells support long term proliferation and differentiation of myeloid and megakaryocytic progenitors. Blood 1995;86: Chute JP, Saini AA, Kampen R et al. A comparative study of the cell cycle status and primitive cell adhesion molecule profile of human CD34 + cells cultured in stroma-free versus porcine microvascular endothelial cell cultures. Exp Hematol 1999;27: Chute JP, Saini A, Wells M et al. Preincubation with endothelial cell monolayers increases gene transfer efficiency into human bone marrow CD34(+)CD38(-) progenitor cells. Hum Gene Ther 2000;11: Brandt JE, Bartholomew AM, Fortman JD et al. Ex vivo expansion of autologous bone marrow stem cells with porcine microvascular endothelial cells results in a graft capable of rescuing lethally irradiated baboons. Blood 1999;94: Shultz L, Schweitzer P, Christianson S et al. Multiple defects in innate and adaptive immunologic function in NOD/LtSzscid mice. J Immunol 1995;154: Trischmann T, Schepers K, Civin C. Measurement of CD34 + cells in bone marrow by flow cytometry. J Hematother 1993;2: van Hennik P, de Koning A, Ploemacher R. Seeding efficiency of primitive human hematopoietic cells in nonobese diabetic/severe combined immune deficient mice: implications for stem cell frequency assessment. Blood 1999;94: