Original Article

Bone Marrow Transplantation (2010) 45, 1000–1007; doi:10.1038/bmt.2009.289; published online 19 October 2009

Cell Selection

CD3+ and/or CD14+ depletion from cord blood mononuclear cells before ex vivo expansion culture improves total nucleated cell and CD34+ cell yields

H Yang1,5, S N Robinson1,5, J Lu1, W K Decker1, D Xing1, D Steiner1, S Parmar1, N Shah1, R E Champlin1, M Munsell2, A Leen3, C Bollard3, P J Simmons4 and E J Shpall1

  1. 1Department of Stem Cell Transplantation and Cellular Therapy, University of Texas MD Anderson Cancer Center, Houston, TX, USA
  2. 2Department of Biostatistics, University of Texas MD Anderson Cancer Center, Houston, TX, USA
  3. 3The Baylor College of Medicine Center for Cell and Gene Therapy, Houston, TX, USA
  4. 4The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston, Houston, TX, USA

Correspondence: Dr SN Robinson, Department of Stem Cell Transplantation and Cellular Therapy, University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Unit 65, Houston, TX 77030-4009, USA. E-mail: snrobins@mdanderson.org

5These authors contributed equally to this work.

Received 3 July 2009; Revised 28 August 2009; Accepted 3 September 2009; Published online 19 October 2009.

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Abstract

Cord blood (CB) is used increasingly in transplant patients lacking sibling or unrelated donors. A major hurdle in the use of CB is its low cell dose, which is largely responsible for an elevated risk of graft failure and a significantly delayed neutrophil and platelet engraftment. As a positive correlation has been shown between the total nucleated cell (TNC) and CD34+ cell dose transplanted and time to neutrophil and platelet engraftment, strategies to increase these measures are under development. One strategy includes the ex vivo expansion of CB mononuclear cells (MNC) with MSC in a cytokine cocktail. We show that this strategy can be further improved if CD3+ and/or CD14+ cells are first depleted from the CB MNC before ex vivo expansion. Ready translation of this depletion strategy to improve ex vivo CB expansion in the clinic is feasible as clinical-grade devices and reagents are available. Ultimately, the aim of improving TNC and CD34+ transplant doses is to further improve the rate of neutrophil and platelet engraftment in CB recipients.

Keywords:

cord blood; ex vivo expansion; inhibitory ‘accessory’ cells; mononuclear cells

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Introduction

Umbilical cord blood (CB) is rapidly becoming an important source of tissue for hematopoietic transplantation largely because of its ethnic diversity, relative ease of collection, ready availability from frozen banks, reduced incidence and severity of GVHD, and tolerance of higher degrees of HLA disparity between donor and recipient when compared with the use of bone marrow (BM) or cytokine-mobilized peripheral blood (PB).1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12

Outside pediatric transplantation, low cell dose remains a major drawback in the successful use of CB transplantation and is largely responsible for the significantly delayed neutrophil and platelet engraftment and an elevated risk of graft failure observed in CB recipients when compared with BM and PB transplant recipients. A positive correlation between the total nucleated cell (TNC) dose7, 11 and CD34+ cell dose12 transplanted and time to neutrophil and platelet engraftment has been identified in CB recipients. To this end, a number of strategies to overcome the low TNC and CD34+ doses associated with CB have been developed, with the aim of improving the speed of neutrophil and platelet engraftment. Approaches towards this currently include the transplantation of two unmanipulated CB units3, 13, 14 and the use of ex vivo expansion techniques.15, 16, 17, 18, 19, 20

Initial ex vivo expansion studies revealed that the selection of CD34+ cells from CB mononuclear cells (MNC) was first required, before successful ex vivo expansion in culture medium containing a cocktail of growth factors was achieved.21 These data suggested that the presence of non-CD34+ ‘accessory’ cells in CB MNC may negatively affect the efficacy of ex vivo expansion. Although CD34+ cell selection from CB MNC was required, it is not an efficient process and significant CD34+ cell losses occur. In the MD Anderson Cancer Center double CB expansion trial, the recovery of CD34+ cells from CB units after positive selection was only 35% (range 4–70%) (EJ Shpall, personal communication). As a direct consequence of the significant CD34+ cell losses, even if significant levels of ex vivo expansion are achieved, cell yields for transplant remain low. Ex vivo expansion strategies that do not require initial CD34+ cell selection were therefore analyzed. It was subsequently shown that ex vivo expansion of primitive and more mature hematopoietic progenitors is achieved by the co-culture of non-selected CB MNC in a culture medium containing a cocktail of growth factors together with cellular and extracellular components of the hematopoietic microenvironment provided by BM-derived MSC.16, 19 This ex vivo co-culture strategy led to the development of a double CB clinical trial at the MD Anderson Cancer Center, with patients receiving one unmanipulated CB unit and one ex vivo-expanded CB unit at transplant. The combination of an unmanipulated CB unit together with an ex vivo co-culture expanded CB unit allows significantly higher doses of TNC and CD34+ cells to be transplanted than have ever previously been achieved. An encouraging trend toward more rapid neutrophil and platelet engraftment has been observed in patients receiving these significantly elevated numbers of TNC and CD34+ cells and provides the impetus to further refine and improve the current ex vivo co-culture expansion strategy.

Although the ex vivo expansion achieved with CB MNC/MSC co-culture is markedly better than that achieved with CD34+ cell selection and liquid culture,19 it was hypothesized that ex vivo expansion would be further improved if putative inhibitory ‘accessory’ cells present in CB MNC were removed before ex vivo CB MNC/MSC co-culture. Previous reports have cited instances in which T lymphocytes,22, 23 natural killer cells,24, 25 macrophages26, 27 and other less well-defined ‘accessory’ cells28, 29 have been shown to negatively affect hematopoiesis. Phenotypic analysis of CB revealed that the major cellular components are CD3+, CD14+, CD56+ and CD19+ cells, and each was analyzed (alone and in combination) with respect to their potential as inhibitory ‘accessory’ cells influencing the magnitude of CD34+ ex vivo expansion achieved.

Initially, using an ‘add-back’ strategy, we showed that the combination of CD3+ and CD14+ cells had a marked inhibitory effect on ex vivo CD34+ expansion, suggesting that CD3+ and/or CD14+ cells were putative inhibitory ‘accessory’ cells. We subsequently confirmed these ‘add-back’ data by a more clinically applicable depletion strategy and showed that ex vivo CD34+ cell expansion was markedly improved after CD3+ and/or CD14+ cell depletion. It is hypothesized that improving the level of ex vivo expansion achieved will increase the TNC and CD34+ cell dose available for transplant and thereby improve platelet and neutrophil engraftment for CB recipients.

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Materials and methods

Cord blood units

Frozen CB units were released for research use under the University of Texas MD Anderson Cancer Center Internal Review Board-approved protocol LAB03-0796.

Use of magnetic-activated cell sorting (MACS)

MACS was used to positively select (for preliminary ‘add-back’ experiments) or deplete (for subsequent depletion experiments) specific cell populations. Single frozen CB units were thawed and washed in CliniMACS wash buffer: CliniMACS buffer (Miltenyi Biotec, Auburn, CA, USA) containing 0.5% human serum albumin (Flexbumin, Baxter Healthcare Corp., Deerfield, IL, USA). Small cell aggregates were removed using sterile 70μm nylon cell strainers (BD Falcon, Bedford, MA, USA). Equal fractions of CB MNC were incubated at +4°C for 5min in a solution of Gammaguard (Baxter Healthcare Corp.) to block nonspecific binding of MACS beads, and at +4°C for 20min in an excess of specific MACS reagents (beads) to positively select CD34+, CD3+, CD14+, CD19+ or CD56+ cells (Miltenyi Biotec). In positively selecting a specific cell population from CB MNC (for example, for use in ‘add-back’ experiments), a product depleted of that specific cell population was also generated (for example, for use in depletion experiments). CD3 and CD14 depletion was achieved by combining CD3 and CD14 MACS beads with CB MNC during the incubation. This simultaneous depletion was shown to be as effective as a two-step, sequential depletion process. A fraction of CB MNC equivalent to that subjected to MACS (selection or depletion) was untreated and served as the unmanipulated CB MNC control.

After incubation, unbound MACS beads were removed by washing in CliniMACS wash buffer. Cells were passed through pre-separation filters (Miltenyi Biotec) and onto MACS LS separation columns according to the manufacturer's instructions (Miltenyi Biotec). LS columns provided both the ‘selected’ product (positive fraction retained in the column and used in ‘add-back’ experiments) and ‘depleted’ product (negative fraction as ‘flow through’ used in depletion experiments). Positive and negative fractions were collected, centrifuged and the cells resuspended in medium. Cell numbers were determined and flow cytometry (see below) was performed to determine the efficiency of the selection/depletion procedure.

‘Add-back’ experiments

‘Add-back’ experiments were designed to expose selected CD34+ cells to the numbers of ‘accessory’ cells’ that would be present in the unmanipulated CB MNC. As five cell populations were required (CD34+, CD3+, CD14+, CD19+ or CD56+ cells), 20% fractions of the total CB MNC were subjected to CD34+, CD3+, CD14+, CD19+ or CD56+ cell selection. CD34+, CD3+, CD14+, CD19+ or CD56+ cells derived from the 20% CB fractions were collected in 2ml of expansion medium (CellGro SCGM (CellGenix, Antioch, IL, USA) containing 10% fetal bovine serum (Hyclone, Logan, UT, USA), antibiotics (100U/ml penicillin and 100μg/ml streptomycin (Gibco, Grand Island, NY, USA) and 2mM L-glutamine (HyClone)), and supplemented with 100ng/ml each of SCF, Flt-3 ligand, TPO (CellGenix) and G-CSF (Neupogen Filgrastim, Amgen, Thousand Oaks, CA, USA). Sixteen different culture combinations were identified: CD34+ cells were cultured alone and with various CD3+±CD14+±CD19+±CD56+ combinations (see Figure 1). Cultures were performed in six-well, flat-bottomed tissue culture plates containing pre-established BM-derived MSC monolayers. MSC were provided as mesenchymal precursor cells (MPC) by Angioblast Systems, Inc. (New York, NY, USA) Ex vivo expansion cultures are routinely performed with 10% of an unmanipulated CB MNC in 50ml of expansion medium in 150cm2 tissue culture flasks containing pre-established monolayers of MSC. With reduction in the size of the culture (culture area reduced from 150cm2 to approximately 7cm2), the number of cells plated (reduced from 10 to 0.5% per culture) and culture volume (reduced from 50 to 2.5ml) were similarly reduced.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Representative ‘add-back’ experiment. CD34+, CD3+, CD14+, CD56+ and CD19+ cells were isolated from cord blood (CB) mononuclear cells (MNC) using magnetic-activated cell sorting (MACS). CD34+ cells were then cultured alone or in the presence of CD3+±CD14+±CD56+±CD19+ cells at levels comparable with those present in the initial CB MNC. Although CD34+ ex vivo cell expansion was reduced in the presence of CD3+ or CD14+ cells, maximal reduction was observed when CD3+ and CD14+ cells were present (indicated by arrows).

Full figure and legend (91K)

Positively selected CD34+ cells derived from 0.5% CB MNC, corresponding to approximately 100μl of the 2ml containing the selected product from 20% of CB MNC, were added to each culture well. For the addition of ‘accessory’ cells, approximately 100μl of the 2ml of cell suspension containing the selected product from 20% of CB MNC (CD3+, CD14+, CD19+ or CD56+ cells) was added to yield the 16 different conditions assayed (CD34+±CD3+±CD14+±CD19+±CD56+).

The expansion medium was added to a final volume of approximately 2.5ml. ‘Add back’ culture was performed at 37°C in a fully humidified atmosphere of 5% CO2 in air. After 7 days, 2.5ml of medium containing non-adherent cells was removed and replaced with 2.5ml of fresh expansion medium and culture was continued for an additional 7 days. In addition, a known volume of the medium containing non-adherent cells was diluted in the fresh expansion medium and culture continued for 7 days in a tissue culture flask at 37°C in a fully humidified atmosphere (‘liquid’ culture) of 5% CO2 in air. After 14 days, all hematopoietic cells (from the ‘liquid’ culture fraction and from the non-adherent and adherent fractions of the co-cultures) were analyzed for TNC number and the number of CD34+ cells present for each condition. The number of hematopoietic cells in the adherent fraction (adhering to the MSC) consistently constituted only a small fraction of the hematopoietic cells in the culture well at day 14.

TNC number was determined using a hemacytometer and the proportion of CD34+ cells present was determined using flow cytometry. Total output TNC and CD34+ numbers were calculated for each condition (CD34+±CD3+±CD14+±CD19+±CD56+). The total output TNC and CD34+ numbers were calculated for each condition and the fold increase in TNC and CD34+ cell numbers from those input (day 0) to those output after 14 days of ex vivo expansion was calculated.

‘Depletion’ experiments

Negative fractions (cells not retained in the MACS column) were collected in 2ml of expansion medium as (1) CD3, (2) CD14 or (3) CD3 and CD14 ‘depleted’ products. As equal fractions of CB MNC were used for selection, all the negative fractions contained similar numbers of the non-depleted populations, that is, CD34+, CD56+ and CD19+ cells. When present, they also contained similar numbers of CD3+ cells (CD14-depleted sample) or CD14+ cells (CD3-depleted sample), or neither (CD3- and CD14-depleted sample). This was confirmed using flow cytometry (data not shown). Cultures were performed in 75cm2 tissue culture flasks (BD Falcon) containing BM-derived MSC monolayers prepared for co-culture, as previously described.19 As the size of the culture was reduced (culture area reduced from 150 to 75cm2), the number of cells cultured and the volume of the incubation medium were similarly reduced.

The depletion product (CD3±CD14 depleted) from approximately 5% CB MNC was cultured in 25ml of expansion medium. The cells were cultured at 37°C in a fully humidified atmosphere of 5% CO2 in air for 7 days. After 7 days, 25ml of medium containing non-adherent cells was removed and replaced with 25ml of fresh expansion medium and culture was continued for an additional 7 days. In addition, a known volume of the medium containing non-adherent cells was diluted in fresh expansion medium and culture of this fraction continued for 7 days in a tissue culture flask at 37°C in a fully humidified atmosphere (‘liquid’ culture) of 5% CO2 in air. At day 14, all hematopoietic cells (from the ‘liquid’ culture fraction and from the non-adherent and adherent fractions of the co-cultures) were analyzed for TNC number and the number of CD34+ cells present for each condition. TNC number was determined using a hemacytometer and the proportion of CD34+ cells present was determined using flow cytometry. Total output TNC and CD34+ numbers were calculated for each condition (MNC or CD3+ and/or CD14+ depleted). Total output TNC and CD34+ numbers were calculated for each condition and the fold increase in TNC and CD34+ cell numbers from those input (day 0) to those output after 14 days of ex vivo expansion was calculated.

Flow cytometric analysis

The cellular composition of the CB MNC before and after MACS selection/depletion, and after culture was confirmed by flow cytometry using fluorescently conjugated detection antibodies (CD34, CD3 CD14, CD19, CD56 and CD45—all BD Biosciences, San Jose, CA, USA). In brief, the cells were washed in flow stain buffer: Dulbecco's phosphate-buffered saline (Gibco) containing 1% (v/v) donor goat serum (JRH Biosciences, Lenexa, KS, USA) and incubated with appropriate fluorescently labeled antibodies for 20min with occasional mixing. Unbound antibodies were removed by washing in flow stain buffer and cells were fixed using a solution of 1.6% (v/v) paraformaldehyde in flow stain buffer. Fluorescence staining was revealed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) and analysis was performed using CELLQuest Pro software (Becton Dickinson). The absolute number of each cell type revealed using flow cytometry (CD34+, CD3+, CD14+, CD19+ or CD56+) was calculated with reference to the appropriate TNC count performed using a hemacytometer.

Statistical analysis

Where appropriate, data were compared using Student's t-test (Excel, Microsoft), with significance assumed at Pless than or equal to0.05.

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Results

‘Add-back’ experiments

Although representative data from a single ‘add-back’ experiment are shown (Figure 1), a similar pattern of ex vivo expansion was observed in other ‘add-back’ experiments. In this representative experiment, CD34+ ex vivo expansion under 16 different conditions is shown. Culture conditions range from CD34+ cells alone, to a combination of cells that was essentially that of CB MNC before any manipulation.

Maximal CD34+ ex vivo expansion (approximately 58-fold) was observed when CD34+ cells were cultured with BM-derived MSC in the expansion medium in the absence of any CB MNC ‘accessory’ cells. This level of CD34+ ex vivo expansion is not markedly different from that observed when CD34+ cells are incubated with CD56+ or CD19+ cells, suggesting that neither of these cell types acting singly inhibit CD34+ ex vivo expansion.

However, in the presence of CD3+ or CD14+ cells, CD34+ ex vivo expansion is reduced (by approximately 50 and 30%, respectively). These data suggest that both of these cell types acting singly inhibit CD34+ ex vivo expansion. Consistent with these data, ‘add-back’ combinations that contain CD3+ or CD14+ cells show a similar trend toward inhibition of CD34+ ex vivo expansion.

However, marked inhibition (>90%) of CD34+ ex vivo expansion was most evident when both CD3+ and CD14+ cells are present (as indicated by arrows in Figure 1). The presence of both CD3+ and CD14+ cells was associated with a profound inhibition of CD34+ ex vivo expansion, be it as part of a two-, three- or four-component ‘add-back’ combination.

Although these data were focused on identifying inhibitory ‘accessory’ cells, no clear evidence for the existence of stimulatory ‘accessory’ cells (acting singly or in combination) was found. CD34+ ex vivo expansion only matched, or was less than that observed with CD34+ cells alone. Evidence of stimulatory ‘accessory’ cells would have been observed as levels of CD34+ ex vivo expansion that exceeded those obtained after culture of CD34+ cells alone.

Efficacy of MACS depletion

Representative flow cytometric data demonstrating the efficacy of depletion using MACS are shown (Figure 2). Consistently high levels of CD3 and/or CD14 depletion were achieved. These data show the specificity and efficacy of the CD3 and/or CD14 depletion process. In this representative sample, the CB MNC contained approximately 56% CD3+ cells and approximately 9% CD14+ cells. After CD3+ and/or CD14+ cell depletion procedures, each population was reduced to 0%. In addition, analysis confirmed that the depletion of CD3+ cells did not affect the numbers of CD14+ cells and vice versa, nor did the depletion of CD3+ and/or CD14+ cells affect the numbers of CD34+ cells. Similarly, no effect on CD19+ and CD56+ cells was observed (data not shown).

Figure 2.
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Efficacy of CD3+ and/or CD14+ depletion using magnetic-activated cell sorting (MACS). Representative flow cytometric analyses of depleted and non-depleted cord blood (CB) mononuclear cell (MNC) products are shown. The efficacy of MACS depletion is shown by the depletion of CD3+ (lower left panel), CD14+ (upper right panel) and CD3+ and CD14+ (lower right panel).

Full figure and legend (119K)

To analyze whether ‘processing’ of the CB MNC for MACS had any effect on the initial input numbers of CD34+ cells and the level of ex vivo CD34+ and TNC expansion achieved, (1) untreated MNC (not exposed to Miltenyi reagents) were passed through pre-separation filters and MACS LS separation columns and (2) CB MNC were treated with either CD3 and/or CD14 reagents and passed through pre-separation filters and MACS LS separation columns that were outside the magnetic separation device. No evidence that such MACS ‘processing’ had any effect on ex vivo expansion was observed (data not shown).

Ex vivo expansion of TNC and CD34+ after CD3+ and/or CD14+ depletion by MACS

The fold expansion of TNC and CD34+ numbers with no depletion (MNC) and after CD3 and/or CD14 depletion (MNC−CD3, MNC−CD14 and MNC−(CD3 and CD14)) is shown in Figures 3 and 4, respectively. (Data are presented as mean fold increase±s.e.m. (n=4–6 replicate experiments).) Control data from CD56-depleted samples are shown for comparison.

Figure 3.
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Effect of CD3+ and/or CD14+ depletion on total nucleated cell (TNC) expansion. TNC numbers are shown for each condition after expansion (mean±s.e.m., n=4–6 replicate experiments. *Statistically significant from mononuclear cells (MNC), Pless than or equal to0.05). Fold increase from input (dashed line) to output is also shown. TNC expansion was approximately 2.3-, 2.2- and 2.1-fold greater than MNC after CD3, CD14 and CD3 and CD14 depletion, respectively.

Full figure and legend (66K)

Figure 4.
Figure 4 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Effect of CD3+ and/or CD14+ depletion on CD34+ cell expansion. CD34+ cell numbers are shown for each condition after expansion (mean±s.e.m., n=4–6 replicate experiments. *Statistically significant from mononuclear cells (MNC), Pless than or equal to0.05). Fold increase from input (dashed line) to output is also shown. CD34+ cell expansion was approximately 2.5-, 2.3- and 2.5-fold greater than MNC after CD3, CD14 and CD3 and CD14 depletion, respectively.

Full figure and legend (61K)

No depletion
 

With no depletion (MNC), an input TNC number of 23.5±2.6 × 106 cells contained approximately 1.3±0.4 × 105 CD34+ cells (approximately 0.6% of pre-expansion TNC). After 14 days of ex vivo expansion culture, TNC and CD34+ cell numbers were increased by approximately 20- and 19-fold, respectively, to 473.5±164.8 × 106 TNC and 24.8±9.8 × 105 CD34+ cells (approximately 0.5% of post-expansion product).

CD3 depletion
 

CD3 depletion (MNC–CD3) reduced the input TNC number from that observed in the non-depleted CB MNC by approximately 42%, from 23.5±2.6 × 106 cells to 13.6±3.3 × 106 cells. The 1.4±0.5 × 105 CD34+ cells contained in the CD3-depleted fraction was similar to the 1.3±0.5 × 105 CD34+ cells contained in the MNC (no depletion). These data show that the depletion of CD3+ cells did not affect the CD34+ population. After 14 days of ex vivo expansion culture, TNC numbers were increased approximately 47-fold from the initial input of 23.5±2.6 × 106 TNC to 1097.3±287.6 × 106 TNC (Figure 3). It should be noted that this expansion actually represents a >80-fold increase over the post-depletion value of 13.6±3.3 × 106 cells.

After 14 days of ex vivo expansion culture, CD34+ cell numbers were increased approximately 48-fold from the initial input of 1.3±0.5 × 105 CD34+ cells to 62.5±24.2 × 105 CD34+ cells (Figure 4). This represents approximately 0.6% of the post-expansion product.

Ex vivo expansion after CD3 depletion delivered significantly greater TNC numbers (2.3-fold, Pless than or equal to0.05) and significantly greater CD34+ cell numbers (2.5-fold, Pless than or equal to0.05) than did ex vivo expansion of unmanipulated CB MNC (no depletion). The increases in TNC and CD34+ cell numbers after CD3 depletion are not significantly different from those obtained after CD14, or CD3 and CD14 depletion.

CD14 depletion
 

CD14 depletion (MNC–CD14) reduced the input TNC number from that observed in the non-depleted CB MNC by approximately 22%, from 23.5±2.6 × 106 TNC to 18.2±1.4 × 106 TNC. The 1.3±0.5 × 105 CD34+ cells contained in the CD14-depleted fraction was identical to that contained in the unmanipulated MNC (no depletion), showing that the depletion of CD14+ cells did not affect CD34+ numbers.

After 14 days of ex vivo expansion culture, TNC numbers were increased approximately 45-fold from the initial input of 23.5±2.6 × 106 TNC to an output of 1097.3±287.6 × 106 TNC (Figure 3). It should be noted that this expansion actually represents a >50-fold increase over the post-depletion value of 18.2±1.4 × 106 TNC. CD34+ cell numbers were increased approximately 44-fold from the initial input to 56.7±25.0 × 105 CD34+ cells (Figure 4). This represents approximately 0.5% of the post-expansion product.

Ex vivo expansion after CD14 depletion delivered significantly greater TNC (2.2-fold, Pless than or equal to0.05) and CD34+ cell numbers (2.3-fold, Pless than or equal to0.05), respectively, than did ex vivo expansion of unmanipulated MNC (no depletion). The increases in TNC and CD34+ cell numbers after CD14 depletion are not significantly different from those obtained after CD3, or CD3 and CD14 depletion.

CD3 and CD14 depletion
 

CD3 and CD14 depletion (MNC–(CD3&CD14)) reduced the input TNC number from that observed in the non-depleted CB MNC by approximately 58%, from 23.5±2.6 × 106 cells to 9.8±1.4 × 106 cells. These data are consistent with the value that would be predicted after the individual depletion of CD3+ (approximately 42%) and CD14+ cells (approximately 22%). The 1.3±0.5 × 105 CD34+ cells contained in the CD3- and CD14-depleted fraction was similar to that present in the unmanipulated MNC (no depletion) and showed that the simultaneous depletion of CD3+ and CD14+ cells did not affect CD34+ numbers.

After 14 days of ex vivo expansion culture, TNC numbers were increased approximately 42-fold from the initial input of 23.5±2.6 × 106 TNC to an output of 982.1±302.7 × 106 TNC (Figure 3).

It should be noted that this expansion actually represents a >100-fold increase from the post-depletion value of 9.8±1.4 × 106 TNC.

CD34+ cell numbers were increased approximately 47-fold from the initial input of 1.3±0.5 × 105 CD34+ cells to 61.5±24.2 × 105 CD34+ cells (Figure 4). This represents approximately 0.6% of the post-expansion product.

Ex vivo expansion after CD3 and CD14 depletion delivered significantly greater TNC (2.1-fold, Pless than or equal to0.05) and CD34+ cell numbers (2.5-fold, Pless than or equal to0.05), respectively, than did ex vivo expansion of unmanipulated MNC (no depletion) when initial (pre-depletion) inputs were compared. The increases in TNC and CD34+ cell numbers after CD3 and CD14 depletion are not significantly different from those obtained after CD3 or CD14 depletion.

CD56+ depletion
 

In a limited number of experiments, CD56 depletion was performed as a control. ‘Add-back’ experiments suggested that the presence of CD56+ cells did not inhibit CD34+ ex vivo expansion (Figure 1). CD56 depletion (MNC−CD56) reduced the input TNC number from that observed in the non-depleted CB MNC by approximately 14% from 23.5±2.6 × 106 cells to 20.2±4.0 × 106 cells. The number of CD34+ cells contained in the CD56-depleted fraction was similar to the 1.3±0.4 × 105 CD34+ cells present in the unmanipulated MNC (no depletion), suggesting that the depletion of CD56+ did not affect CD34+ numbers.

After 14 days of ex vivo expansion culture, TNC numbers were increased approximately 18-fold from the initial input of 23.5±2.6 × 106 TNC to an output of 416.7±187.7 × 106 TNC (Figure 3).

It should be noted that this increase in TNC numbers actually represents a >20-fold increase from the post-depletion value of 20.2±4.0 × 106 TNC. CD34+ cell numbers were increased approximately 14-fold from the initial input of 1.3±0.5 × 105 CD34+ cells to 18.1±1.4 × 105 CD34+ cells (Figure 4). This represents approximately 0.4% of the post-expansion product.

The increase in TNC and CD34+ cell numbers after CD56 depletion is significantly less than that obtained after CD3 and/or CD14 depletion (Pless than or equal to0.05); however, it is not significantly different (P>0.05) from that obtained after the ex vivo expansion of unmanipulated (non-depleted) MNC.

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Discussion

Strategies to overcome the low TNC and CD34+ doses associated with CB transplantation have been developed with the aim of improving the speed of neutrophil and platelet engraftment and reducing the risk of graft failure, and include the transplantation of two unmanipulated CB units3, 13, 14 and the ex vivo expansion of one of the two CB units before transplant.15, 16, 17, 18, 19, 20 In this study we focus specifically on the contribution of ex vivo CB expansion and show that a relatively simple refinement to a current ex vivo CB MNC/MSC co-culture expansion technique might further improve the magnitude of ex vivo expansion achieved and therefore possibly positively affect the rate of neutrophil and platelet engraftment and reduce the risk of graft failure.

Ex vivo expansion using a liquid culture technique was shown to be effective only if CD34+ or CD133+ CB progenitors were first positively selected from the CB MNC cell milieu21 (even though an inefficient process), suggesting that the removal of the putative negative influences of non-CD34+ or non-CD133+ ‘accessory’ cells was required. In this study we present evidence for the presence of negative ‘accessory’ cells in CB MNC and show that the depletion of these inhibitory ‘accessory’ cells and/or the factors they produce improves ex vivo expansion in a CB MNC/MSC co-culture system.

On the basis of ‘add-back’ experiments, CD3+ and CD14+ cells seemed to be candidate inhibitory ‘accessory’ cells. Subsequently, ex vivo expansion of CD34+ in CB MNC/MSC co-culture was performed, in which CD3+ and/or CD14+ cells were first depleted from the CB MNC. We show that the depletion of CD3+ and/or CD14+ cells improves the ex vivo expansion of TNC and CD34+ cells obtained with CB MNC/MSC co-culture.19

‘Add-back’ experiments showed that both CD3+ and CD14+ cells were required before a maximal negative effect on CD34+ ex vivo expansion was observed. These data were confirmed by the observation that CD34+ cell expansion was improved (>twofold) with the depletion of both CD3+ and CD14+ cells from CB MNC before CB MNC/MSC co-culture. However, a similar improvement in CD34+ cell expansion was also observed when either CD3+ or CD14+ cells were depleted from CB MNC before MSC co-culture. These data suggest that the removal of one or other or both populations has a similar positive effect on CD34+ ex vivo cell expansion. This observation might be explained by the possible existence of an inhibitory ‘cross-talk’ between the CD3+ and CD14+ cell populations. One population may stimulate the production of an inhibitory activity by the other to ultimately regulate CD34+ proliferation. Alternatively, each population may produce unique factors that may synergize to inhibit CD34+ ex vivo expansion. In such models, the removal of one or other or both populations could therefore be explained to similarly improve CD34+ ex vivo expansion.

Targeting the depletion of one inhibitory ‘accessory’ cell population (CD3+ or CD14+ cells) rather than both (CD3+ and CD14+ cells) to improve ex vivo CB expansion reduces both the complexity and cost of the procedure. In addition, there may be advantages to the targeted depletion of only one population. For example, the CD3+ cell population collected after CD3 depletion could be used to further develop immunological and/or anti-tumor strategies to potentially supplement hematopoietic reconstitution of the transplant recipient.30, 31, 32

In addition, the observation that inhibitory activity may be associated with CD14+ cells present in the CB MNC might not be surprising as macrophages have previously been shown to produce stem cell-specific proliferation regulators.33, 34, 35, 36 Whether the putative CD3+/CD14+ interaction requires cell-to-cell contact, and/or is mediated through humoral factors, is under investigation. Further, the specific subsets of the CD3+ population and/or the CD14+ population, which may be responsible for the inhibitory activity, remain to be identified.

The prospect of identifying possible ‘inhibitory factors’ lends itself to the possible clinical application of activity-neutralizing antibodies and/or function-blocking agents to optimize ex vivo CD34+ expansion. Such an approach may replace the need for depletion to remove specific ‘accessory’ cells.

The inhibition of CD34+ ex vivo cell expansion observed in the presence of both CD3+ and CD14+ cells during CB MNC/MSC co-culture might only be a relatively short-lived early event, as the current ex vivo culture conditions (SCF, TPO, G-CSF and Flt-3L) do not promote CD3+ cell survival. CD3+ cells are therefore ‘lost’ from the culture relatively early in the ex vivo cell expansion culture, presumably releasing their contribution to any putative inhibition of CD34+ ex vivo expansion. This would restrict the effect of any CD3+/CD14+ inhibitory ‘cross-talk’ on the earliest parts of the CB MNC/MSC co-culture. This might, at least in part, explain the initial ‘lag’ observed in ex vivo cell expansion cultures. It is possible that the greater-than-twofold improvement in TNC and CD34+ ex vivo expansion observed with CD3+ and/or CD14+ depletion is a consequence of reducing this initial ‘lag’ in TNC and CD34+ ex vivo expansion.

The attraction of a CD3+ or CD14+ depletion strategy to remove inhibitory ‘accessory’ cells and/or the factors they produce from the CB MNC/MSC co-culture and improve CD34+ expansion is that it does not adversely affect the input CD34+ population, unlike the use of a positive CD34+ selection strategy in which significant CD34+ losses are incurred. In addition, the relative ease, rapidity and availability of clinical-grade Food and Drug Administration-compliant devices and MACS reagents (Miltenyi Biotec) give this approach a significant advantage over other selection/depletion techniques. As clinical-grade devices and reagents are available, the use of an ‘accessory’ cell depletion strategy and its beneficial effect on ex vivo CB expansion could be readily translated to the clinic. Ultimately, increased TNC and CD34+ cell doses may contribute to improve neutrophil and platelet engraftment in CB recipients, thereby improving the efficacy and ultimately the cost effectiveness of CB transplantation as a treatment rationale.37

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Conflict of interest

The authors declare no conflict of interest.

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Acknowledgements

This work was supported by NCI R01 CA061508-16. The authors gratefully acknowledge the provision of MPC by Angioblast Systems, Inc., New York, NY.