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Defining Optimum Conditions for the Ex Vivo Expansion of Human Umbilical Cord Blood Cells. Influences of Progenitor Enrichment, Interference with Feeder Layers, Early-Acting Cytokines and Agitation of Culture Vessels

^a Universitätsklinikum Carl Gustav Carus, Med. Klinik I, Dresden, Germany;
^b In-vitro Systems GmbH, Osterode, Germany

Key Words. Cord blood • Expansion • Feeder layer • MGDF • CD34 selection

Correspondence: Dr. Martin Bornhäuser, Medical Clinic I, University Hospital Carl Gustav Carus, Fetscherstraße 74, 01307 Dresden, Germany.

	Abstract

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Ex vivo expansion of human umbilical cord blood cells (HUCBC) is explored by several investigators to enhance the repopulating potential of HUCBC.

We performed experiments using either Ficoll-separated or CD34⁺-selected HUCBC from the same donation in serum-free medium. CD34-purified HUCBC were cultured on either human umbilical vein endothelial cells (HUVEC) or irradiated bone marrow-derived stroma cells (BMSC) with addition of different cytokines. In addition, we tested the expansion of HUCBC in culture vessels with continuous rotation.

CD34 enrichment led to a significant increase in the expansion factor of CD34⁺ cells compared with unmanipulated HUCBC. BMSC were more efficient in amplifying early progenitors than HUVEC. Optimum results were reached by a combination of SCF, FLT-3L at 300 ng/ml and IL-3 at 50 ng/ml. No significant improvement in the expansion of CD34⁺/38^– primitive progenitors could be obtained with other combinations. Addition of megakaryocyte-derived growth and development factor to each growth factor cocktail improved the expansion results. Continuous rotation of culture vessels did not ameliorate the expansion rate of the analyzed subsets. Culture conditions separating stroma and HUCBC by a semipermeable membrane improved the expansion factors of CD34⁺, CD34⁺/38^–, and CD34⁺/41⁺ cells and CFU-GM compared with contact cultures. These data might be useful when designing culture systems for clinical scale ex vivo expansion of HUCBC.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Human umbilical cord blood cells (HUCBC) have become an attractive source of hematopoietic precursors for allogeneic blood stem cell transplantation in children with inborn errors or malignant diseases [1, 2]. One major advantage of HUCBC in comparison with peripheral blood stem cells (PBSC) or bone marrow (BM) might be the reduced incidence of acute graft-versus-host disease caused by cord blood grafts [3]. Although HUCBC are easy to collect and store, the introduction into transplantation protocols for adults has been hampered by the limited number of progenitors contained in one cord blood harvest. The prolonged time to engraftment as well as the existence of very early hematopoietic progenitors have led hematologists to explore ex vivo expansion of HUCBC for clinical use [4].

Optimal consistence of different media as well as different growth factor cocktails have been evaluated in detail [5, 6]. Those experiments resulted in efficient expansion of progenitors, but long-term culture initiating cells (LTC-IC) or mouse-repopulating cells have not been amplified convincingly thus far. There are several studies indicating the importance of medium changes or perfusion of culture vessels as well as stirred conditions for enhanced expansion rates [7, 8]. Clinical scale bioreactors have been designed and tested in phase I studies in patients receiving expanded BM or PBSC after nonmyeloablative conditioning therapies [9].

Intermittent interactions of HUCBC with stromal cells, elimination of metabolites from the culture medium, oxygenation and pH have been shown to be independent factors which must be controlled in bioreactor systems [10]. In a series of experiments, we tried to define optimal conditions for culture of HUCBC in static culture conditions. We tested the influences of CD34 enrichment, different feeder layers, and megakaryocyte-derived growth and development factor (MGDF) on cell expansion kinetics. Additional experiments were performed to investigate the role of physical stromal contact by introduction of semipermeable membranes into culture dishes. Effects of continuous rotation of culture vessels were evaluated and compared with those of static cultures.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell Separation
Cord blood was collected and diluted with phosphate-buffered saline (PBS) Dulbecco's without magnesium and calcium, pH 7.2 (GIBCO; Paisley, Scotland). Density gradient centrifugation was performed as follows: cells were centrifuged with Immuflot (Immucor GmbH; Rödermark, Germany) at 800 g for 20 min, mononuclear cells were collected and washed twice with PBS, supplemented with 0.5% human serum albumin ([HSA] 5%, Immuno GmbH; Heidelberg, Germany). Pellets were resuspended in 5 ml of CellGro^® SCGM ([stem cell growth medium], Boehringer Ingelheim Bioproducts; Mannheim, Germany). Cells were counted automatically by Technicon H 3 RTC^TM (Bayer Diagnostics GmbH; München, Germany).

In some cases, lysis of red cells in HUCB was tested. About 50 ml of cord blood were mixed with 200 ml of ice-cold lysis-buffer, consisting of 8.29 g ammonium chloride, 1.00 g potassium hydrogen carbonate, and 0.037 g sodium-EDTA in one liter aqua with a pH of 7.4. After 15 min of incubation, the nucleated cells were separated by centrifugation, supernatant was removed, and the pellet was resuspended in 5 ml of CellGro^® SCGM (Boehringer Ingelheim).

Establishment of Stromal Layers
Bone marrow aspirates were obtained from healthy donors after informed consent. The marrow was mixed with the same volume of PBS, and mononuclear cells were separated by density gradient centrifugation. Washed cells were resuspended in stroma medium (RPMI 1640, Dutch Modification, supplemented with 10% fetal calf serum [FCS], 2 mM L-glutamine, 100 IU/ml penicillin, 0.1 mg/ml streptomycin, and 1.0 µmol/l hydrocortisone).

5 x 10⁶ cells were placed in 80-cm² flasks (Nunc; Wiesbaden, Germany) and maintained at 37°C and 5% CO₂. Half the medium was exchanged twice a week. After two weeks, confluent adherent layers were passaged with trypsin-EDTA solution 1X (Sigma-Aldrich; Steinheim, Germany). Trypsin was quickly inactivated with FCS-containing medium and centrifuged at 200 g for 10 min. The pellet was resuspended in stromal medium, and the cells of one 80-cm² culture vessel were transferred in six 25-cm² flasks or six six-well plates for further expansion experiments. Confluent layers were irradiated with 15 Gy before initiating culture experiments.

Establishment of HUVEC
Umbilical cords were placed in a PBS solution containing 10 mg/ml gentamycin, 250 µg/ml amphotericin B, and 5,000 U/ml sodium-heparin (Sigma-Aldrich). The umbilical cord vein was punctured and flushed with PBS several times. Collagenase solution 0.05% (Sigma-Aldrich) was instilled and incubated for 4 min at 37°C. To stop the enzymatic reaction, trypsin inhibitor solution from soybean was used. Residual cells were harvested by flushing the vein with PBS again.

An additional centrifugation step and resuspension of the cells in endothelial cell growth medium ([ECGM], PromoCell; Heidelberg, Germany) with a low content of FCS followed. HUVEC were placed in a culture flask coated with fibronectin (Sigma; 20 µg/ml) and maintained at 37°C and 5% CO₂.

Half the medium was exchanged three times a week until confluence was reached. Treatment with trypsin and further maintenance of the cultures was performed as mentioned above.

CD 34⁺ Progenitor Cell Purification
HUCBC were enriched using the MACS system (CD34 Selection Kit, Miltenyi Biotec GmbH; Bergisch-Gladbach, Germany) according to the manufacturer's instructions. Briefly, HUCBC were washed and resuspended in PBS, 0.5% bovine serum albumin, and 5 mmol/l EDTA. Cells were first incubated with QBEND-10 antibody (mouse antihuman CD34) in the presence of human IgG as blocking reagent. After one cell wash, another incubation step with 100 µl MACS microbeads per 10⁸ cells followed. Labeled cells were loaded onto a column installed in a magnetic field. Trapped cells were eluted after removal of the column from the magnet.

Methylcellulose Progenitor Culture
As described, 1 x 10⁵ HUCBC or 1 x 10³ CD34-enriched HUCBC were plated in a complete methylcellulose medium containing IMDM with 30% fetal bovine serum, 3 U/ml erythropoietin, 50 ng/ml SCF, 20 ng/ml GM-CSF, 20 ng/ml IL-3, 20 ng/ml IL-6, and 20 ng/ml G-CSF (Methocult GF H4435, Stem Cell Technologies; Vancouver, Canada). Cultures were incubated at 37°C and 5% CO₂. The cultures were assessed at days 12 to 14 for the presence of burst-forming unit-erythroid, colony-forming unit-granulocyte-macrophage, and mixed colony-forming unit.

Expansion Cultures
HUCBC were cultured in 25 cm² flasks containing 10 ml serum-free medium (CellGro^® SCGM, Boehringer Ingelheim) at 37°C and 5% CO₂ for eight days. Preformed stroma or HUVEC were used as feeder-layer, when indicated. Growth factor cocktail 1 contained SCF 300 ng/ml (Amgen; München, Germany), FLT3-ligand 300 ng/ml, and IL-3 50 ng/ml (Genzyme; Mannheim, Germany). Cocktail 2 contained the cytokines of cocktail 1 supplemented with G-CSF 450 ng/l (Amgen), IL-6 100 ng/ml (Genzyme), and erythropoietin 1 U/ml (Genzyme). MGDF (Amgen) was added at concentrations of 25 and 100 ng/ml. Unselected and CD34-enriched HUCBC were cultured at 5 x 10⁵ and 1 x 10⁴/ml, respectively.

Flow Cytometric Analysis
Analysis of the CD34 content of all samples before and after expansion was performed with FACS SCAN (Becton Dickinson; San Jose, CA) using a class III CD34 antibody HPCA-2-PE, (Becton Dickinson) and standard software LYSIS II. Double-color staining and analysis were performed for all samples using the following antibodies: anti-CD41^–FITC, anti-CD38^–FITC (Coulter-Immunotech Diagnostics; Miami, FL), and anti-CD117-PE (c-kit) (Dako Diagnostika; Glostrup, Denmark). Double-staining of CD34/CD38, CD34/CD117, and CD34/CD41 was performed to quantitate progenitor subsets.

To test for viability, propidium iodide (PI) staining was performed after all cultures. Annexin was used to determine the percentage of apoptotic cells. PI^–/Annexin⁺ events defined early apoptosis, whereas PI⁺/Annexin⁺ signals were counted as late apoptosis.

Transwell Cultures
To test the influence of stroma contact, six-well plates (Nunc) were used. The culture dishes were separated by microporous membranes (0.2 or 3 µm) which do not allow cell migration but do allow diffusion of soluble factors like chemokines.

Rotating Cultures
A rotating 50 ml "3D Culture Module" (Heraeus; Osterode, Germany) was compared with static cultures. Culture vessels were maintained at 5% CO₂ and 37°C with a continuous rotation of 2 Upm.

Statistical Analysis
For most parameters, median and range are provided. Experiments evaluating different conditions were compared using the Wilcoxon-signed rank test for paired samples. Statistical significance was assumed when the two-tailed p value was below 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
CD34 Enrichment
CD34 enrichment and low-density culture showed significant enhancement of the expansion factors. Figure 1 compares the expansion factors of CD34⁺ cells with and without prior immunomagnetic enrichment. The median expansion factor of CD34⁺ cells was 4.2 (2.0-8.7) in unselected HUCBC and 29 (15-53) in the CD34-enriched controls (p = 0.03). Interestingly, the experiment with HUCBC at a purity of 96% did not provide the best results. The median expansion factor of total nucleated cells was 1.82 (0.8-3.2) for unselected samples compared with 237 (135-1,345) for CD34-enriched HUCBC (p = 0.016).

View larger version (12K): [in this window] [in a new window]   Figure 1. The bars represent the expansion factors reached in six experiments comparing either CD34 enriched or unselected HUCBC of the same origin. CD34 purity of each starting sample is provided on the x-axis.

View larger version (14K): [in this window] [in a new window]   Figure 2. Influence of feeder layers. The starting cell concentration of CD34-enriched HUCBC was 1 x 104/ml. Median absolute cell counts in 10 ml cultures after 8 days were compared to controls without feeder layer support. The cytokines used in these experiments were SCF and FLT-3L at 300 ng/ml and IL-3 at 50 ng/ml, respectively. HUVECS = human umbilical vein endothelial cells. BMSC = bone marrow-derived stroma cells; NC = nucleated cells.

View larger version (15K): [in this window] [in a new window]   Figure 3. MGDF and other growth factors. A starting cell concentration of 1 x 104/m1 CD34 enriched HUCBC was inocculated. FLT-3L, SCF at 300 ng/ml and IL-3 at 50 ng/ml (cocktail 1) were either supplemented by MGDF at 25 ng/ml (left graph) or with G-CSF 450 ng/ml, EPO 1 U/ml and IL-6 at 100 ng/ml (right graph). The absolute numbers of CD34+, CD34+/38– and CD34+/41+ cells after 8 days of culture are compared.

Rotation
No significant differences in the expansion factors of CD34⁺ subsets and CFU-GM could be found in cultures with continuous rotation at 2 Upm in comparison with static control cultures.

Stroma Contact
Table 1 shows the results of cultures comparing cultures in transwell experiments. The numbers provided are the expansion factors for each subset, respectively.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

There are some reports about the possible role of accessory cells for the expansion of bone-marrow-derived stem cells, which might also be true for the clonogenic potential of early cord blood progenitors [14]. Since we performed CD34 enrichment with one round of immunomagnetic separation, the purity reached (30%-70%) led to a decrease in cell concentrations but still left some cells with possible accessory function, e.g., lymphocytes and monocytes in the primary sample. The debulking of the cord blood product is especially desirable when clinical scale expansion is started and the volume of culture media may increase significantly [15]. Nevertheless, recent reports on the existence of CD34^– long-term repopulating cells have to be kept in mind when CD34⁺ selection is performed [16, 17].

The combination of SCF, FLT-3L, and IL-3 showed the best expansion results. Recently, this combination was described as favorable even for the expansion of SCID repopulating CD34^– stem cells [16].

Like other cytokines, MGDF was found to act not solely on megakaryocytes but also in the expansion of more primitive progenitors, especially when combined with other early-acting cytokines such as Flt3-ligand and SCF [18, 19]. In our experiments, these findings could be confirmed and the increase of CD34⁺/CD41⁺ lineage-determined megakaryocytic progenitors was marked as well.

Taken together, the optimum combination of cytokines in our experiments was SCF, Flt3-ligand, MGDF, and IL-3. Similar growth factor combinations have been shown to conserve the recloning potential of HUCBC in stroma-free cultures for up to 20 weeks [20]. As other authors have shown for bone-marrow-derived progenitors in small [21] and large-scale experiments [22], the addition of EPO, IL-6, and G-CSF tended to improve the expansion results.

Since stroma-derived factors are known to be supportive for the culture of blood stem cells, we tested HUVEC and irradiated BM stroma from healthy volunteers for their influence on the expansion results [23]. As previously speculated, the expansion of total NC, CFU-GM, and CD34⁺ NC was enhanced by culturing on both feeder layers. Nevertheless, BMSC led to a better expansion than HUVEC. This may be due to the more supportive structure of the BM stroma layer and the different soluble chemokines secreted by both types of feeder cells [24]. BM stroma is known to produce SDF-1, which binds to chemokine receptors such as CXCR-4 on early CD34⁺ progenitors [25].

To test the influence of stroma contact on the expansion of umbilical cord blood, we performed cultures in dishes where the HUCBC were separated from the feeder layer by microporous membranes. These experiments showed that no-contact cultures which allow only small molecules to traffic between stroma cells and HUCBC improved the expansion results in terms of total NC, CD34⁺, CD34⁺/38^–, CD34/41⁺ cells, and CFU-GM. Other investigators have confirmed that stroma contact might not be necessary for the expansion of LTC-IC [26, 28]. No comparable data are available for HUCBC. Nevertheless, contact might be necessary to preserve in culture the pluripotent, perhaps even CD34^- stem cell. This pre-stem cell might need intermittent cell-cycle arrest in a niche provided by stroma cells to perform asymmetric division and thereby enabling the hematopoiesis to be regenerated for an individual life span. A recent report states the length of the culture to be a crucial factor in terms of loss of primitive pluripotent stem cells [29].

The culture vessels depicted in the Materials and Methods section which were rotated during the whole culture period were not able to optimize the expansion results in most cases. Even though the permanent agitation may optimize gas exchange and suspension of the media components [8], no corresponding effects in terms of better culture results were measurable in our experiments.

The rate of apoptosis is known to increase in CD34⁺ progenitors during in vitro culture and seems to be a useful marker for the assessment of the quality of stem cell grafts [30]. The increased rate of apoptosis may be associated with proliferation and cell division or induced by the cytokines activating more primitive cells [31]. Some authors have found MGDF and other early-acting cytokines to be able to suppress apoptosis in single-cell experiments [32]. The rate of apoptosis measured by annexin positivity in our bulk cultures was not lowered by adding MGDF. Interestingly, there was a trend toward decreased apoptosis in cultures with bone marrow stroma cells, indicating the supportive character of feeder layers.

The addition of stroma-coated microspheres could be a way to ameliorate the results of rotation cultures, but large-scale expansion with these particles in a GMP-like fashion appears to be a difficult task.

The results obtained in our experiments can lead to the design of a new perfusion bioreactor. A compartment containing a feeder layer has to be separated from HUCBC in suspension culture by a semipermeable membrane, allowing the diffusion of soluble chemokines but avoiding direct contact. This so-called "no-contact system" might be perfused with a serum-free medium containing recombinant GMP-grade cytokines. Whether this integrated concept will lead to an expansion of committed progenitors and a conservation of pluripotent stem cells at the same time has to be awaited.

	Acknowledgments

 
We would like to thank the Deutsche Knochenmarkspenderdatei DKMS for the support. G-CSF, MGDF and SCF used in the experiments were provided by Amgen (München, Germany).

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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