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Stem Cells 2004;22:202-215 www.StemCells.com
© 2004 AlphaMed Press

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. Chutea,b, Garrett Muramotoa, Jennifer Fungc, Carol Oxfordd

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

John P. Chute, M.D., Stem Cell Transplantation Program, Duke University, 2400 Pratt Street, Suite 1100, Durham, North Carolina 27710, USA. Telephone: 919-668-1011; Fax: 919-668-1091; e-mail: johnchute{at}duke.edu


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 HUBEC-secreted soluble factors critically supported SRC self-renewal. 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.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
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 [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
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation of Primary Human Brain Endothelial Cells
Vessel segments (<10 cm) from the central nervous system were collected from cadavers within 12 hours post-mortem under an approved tissue procurement protocol. Vessel segments were placed in complete endothelial cell culture medium containing M199 (GIBCO/BRL; Gaithersburg, MD; http://www.invitrogen.com), 10% heat-inactivated fetal bovine serum (FBS; HyClone Lab; Logan, UT; http://www.hyclone.com), 100 µg/ml L-glutamine, 50 µg/ml heparin, 30 mg/ml endothelial cell growth supplement (Sigma; St. Louis, MO; http://www.sigmaaldrich.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; http://www.allcells.com). HUBECs were subcultured at 1 x 105 cells/well in gelatin-coated 6-well plates (Costar; Cambridge, MA; http://www.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’s-modified 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; http://www.rndsystems.com) in each well. Liquid suspension cultures were performed with the identical cytokines and concentrations. Purified CB CD34+ cells (1 x 105) were added to each well, and cultures were maintained at 37°C in 5% CO2. 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; http://www.bd.com) 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 (5–500 x 102) 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 (1 x 105) 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 {chi}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 {chi}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 5 x 105 fresh CB CD34+ cells, the progeny of 5 x 105 CB CD34+ cells cultured with TSF for 7 days, or the progeny of 5 x 105 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, 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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
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 1Go). As shown in a representative experiment (Fig. 1Go), 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. 1AGo, 1BGo). 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. 1CGo). 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. 1DGo).


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  		Table 1. HUBEC coculture supports greater expansion of human CB progenitor cells
 


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  		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.

 
HUBEC coculture induced a 73-fold increase in colony-forming 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 1Go). 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 2AGo, transplantation of 1 x 103 fresh CB CD34+ cells resulted in no engraftment in eight mice. Similarly, the progeny of 1 x 103 CB CD34+ cells cultured with TSF also failed to engraft in 10 mice. The progeny of 1 x 103 CB CD34+ cells cultured with HUBECs engrafted in 1 of 10 mice (10%) (Fig. 2AGo). Over a dose range of 5 x 103 to 1 x 104 cells, fresh CB CD34+ cells engrafted in only 4 of 16 mice (25%), whereas the progeny of 5 x 103 to 1 x 104 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. 2BGo, 2CGo). Similarly, at a dose of 1 x 104, 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 5 x 104 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 5 x 104 CB CD34+ cells cultured with TSF engrafted in 7 of 8 mice (88%) (Fig. 2DGo).



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  		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) 1 x 103 CB CD34+ cells (left) or their progeny following TSF culture (middle) or HUBEC coculture (right) x 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 5 x 103 CB CD34+ cells or their progeny. C) Human cell engraftment following transplantation of 1 x 104 CB CD34+ cells or their progeny. At the second from right, human cell engraftment is shown in mice transplanted with the progeny of 1 x 104 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 1 x 104 CB CD34+ cells following 14 day coculture with HUBECs. D) Human cell engraftment following transplantation of 5 x 104 CB CD34+ cells or their progeny.

 
In order to assess whether cell-to-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-to-cell contact. As shown in Figure 2CGo, the progeny of 1 x 104 CB CD34+ cells plated in noncontact HUBEC cultures repopulated 8 of 9 mice (89%) and at levels of human 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 1 x 104 CB CD34+ cells following 14 days of HUBEC coculture. As shown in Figure 2CGo, the progeny of day-14 HUBEC cultures repopulated 100% of mice with a 4.2-fold higher 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 14.

HUBEC-Cultured CB Cells Demonstrated Multilineage Differentiation In Vivo
Figure 3Go shows human CD45+ cell engraftment at week 8 in representative mice that were transplanted with fresh CB CD34+ cells (5 x 103) (Fig. 3AGo), the progeny of the same dose of CB CD34+ cells cultured with TSF (Fig. 3BGo), and the same dose of CB CD34+ cells cultured with HUBEC monolayers (Fig. 3CGo). As shown in Figure 3DGo, 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 2Go). 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.



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  		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 5 x 103 CB CD34+ cells. B) Expression of human CD45+ cells in a mouse transplanted with the progeny of 5 x 103 CB CD34+ cells following culture with TSF alone. C) Expression of human CD45+ cells in a mouse transplanted with the progeny of 5 x 103 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.

 

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  		Table 2. HUBEC-cultured cells generate multilineage human cell differentiation in vivo
 
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 {chi}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. 4AGo). The SRC frequency within cells cultured with TSF alone was 1 in 16,000 (CI, 1/9,700- 1/29,000; Fig. 4BGo). The SRC frequency within HUBEC-cultured cells was significantly higher, at 1 in 6,100 (CI, 1/3,700–1/11,000; Fig. 4CGo). 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 = 0.011 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 {chi}2 estimation [36]. Again, the {chi}2 value was not significant in all cases (p > 0.6).



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  		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 5AGo 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 5 x 105 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. 5AGo). These cells were then analyzed for CD45 expression as well as high forward scatter. Figure 5AGo 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.



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  		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 5 x 105 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 5 x 105 CB CD34+ cells cultured with TSF x 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 5 x 105 CB CD34+ cells cocultured with HUBEC x 7 days. A significantly higher percentage of engrafted CD34+CD45+ cells is shown.

 
As shown in Figure 5BGo, 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 5CGo. 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 6AGo, the differences in the engraftment of HUBEC-cultured CB CD34+ cells versus fresh CB CD34+ cells and versus TSF-cultured 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 HUBEC-cultured 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. 6BGo), although these differences were not statistically significant (p > 0.2 for both; Wilcoxon rank sum test).



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  		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 5 x 105 fresh CB CD34+ cells versus the progeny of 5 x 105 CB CD34+ cells cultured with TSF x 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.

 
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 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 = 0.007 and p = 0.02, respectively; Wilcoxon rank sum test). Figure 7Go 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.



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  		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.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
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 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 TSF alone. 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 HUBEC-cultured 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 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 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 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 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 HUBEC-cultured 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 HUBEC-cultured 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 HUBEC-cultured 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 aorta-gonad-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.


   ACKNOWLEDGMENT
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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-97-588 between NMRC and Large Scale Biology Corporation.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received September 5, 2003; accepted for publication October 28, 2003.



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