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Stem Cells, Vol. 17, No. 1, 31-38, January 1999
© 1999 AlphaMed Press

Thrombopoietin Combined with Early-Acting Growth Factors Effectively Expands Human Hematopoietic Progenitor Cells In Vitro

Jan-Arne Hunnestad, Rita Steen, Geir E. Tjønnfjord, Torstein Egeland

Institute of Transplantation Immunology and Medical Department A., The National Hospital, University of Oslo, Oslo, Norway

Key Words. Thrombopoietin • Hematopoietic progenitor cells • CD34 • Expansion • Stem cell transplantation • Megakaryocytopoiesis

Correspondence: Dr. Jan-Arne Hunnestad, Institute of Transplantation Immunology, The National Hospital, N-0027 Oslo, Norway.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Thrombopoietin (TPO) is established as a powerful stimulant of megakaryocyte differentiation and platelet production both in vivo and in vitro. In preparation for future transplantation of ex vivo expanded CD34+ hematopoietic progenitor cells (HPCs), we have examined the in vitro effect of TPO on cultures of HPC when combined with other early-acting hematopoietic growth factors (GFs) in an attempt to decrease post-transplant thrombocytopenia and accelerate engraftment. By adding TPO to all possible combinations of GM-CSF, IL-3, and c-kit ligand (CKL) in a suspension culture system, we found a significant increase in both relative and absolute numbers of cells in cultures containing TPO of the megakaryocytic lineage and CD34+ cells after 14 days of culture.

The most efficient GF combinations for expansion of cell populations of the megakaryocytic lineage and HPCs were TPO, GM-CSF, and CKL, which increased the number of cells of the megakaryocytic lineage 78 fold and the number of CD34+ cells 1.8 fold. The number of CD34+ cells decreased in the cultures containing GM-CSF and CKL with no TPO present, and the number of cells of the megakaryocytic lineage was increased merely 27 fold. Based on our findings, we suggest adding cells from HPCs expanded in cultures containing TPO, GM-CSF, and CKL to unexpanded stem cells for stem cell transplantation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
One of the main side effects of bone marrow transplantation is thrombocytopenia, and most patients have the need for platelet transfusions after transplantation. The ligand for mpl, also labeled thrombopoietin (TPO), was identified and cloned in 1994 [1-4]. Since TPO is a growth factor (GF) that gives high increase in platelet production, there have been several studies to elucidate its effects both in vivo [2, 5-9] and in vitro [10]. The mpl receptor is expressed on megakaryocytic progenitors, at least from the time they coexpress CD61 [11], but it has also been found on more primitive hematopoietic progenitor cells (HPCs) [12]. In animal models, TPO expands cell populations of both the megakaryocytic and erythroid lineage as well as HPCs in general [5-8, 13]. After bone marrow transplantation, the endogenous TPO level is already elevated and the lack of target cells, i.e., megakaryocytes and megakaryocytic lineage cells, makes further addition of TPO less effective than in healthy subjects [9].

Regarding the effect of TPO on HPCs, studies on murine [14, 15] and human [16] HPCs show that TPO induces HPC differentiation into megakaryocytes and proplatelets and stimulates the generation of colony-forming unit-megakaryocytes (CFU-Mks). This effect might be utilized in clinical stem cell transplantation. Among the early-acting cytokines, two of these have been shown to have an expanding effect on the HPC population as well as on cell subsets of the megakaryocytic lineage—GM-CSF and interleukin 3 (IL-3) [15, 17, 18]. Further, c-kit ligand (CKL) is well established as an effective stimulator of HPCs [19, 20] and is known to interact with TPO, GM-CSF, and IL-3 [15]. In these studies, IL-3, GM-CSF, and CKL have been shown to generate only modest numbers of megakaryocytes or platelets in vitro, but combined with TPO an additive or even synergistic effect occurs.

Focusing on a GF combination that could reduce thrombocytopenia and probably also accelerate engraftment after stem cell transplantation, the aim of the present study was to further define the in vitro effect of TPO on HPCs in combination with one or several of the GFs: GM-CSF, IL-3, and CKL. Our results show that TPO has a significant effect on the in vitro generation of megakaryocytic progenitors and more mature megakaryocytic cells as well as HPCs. Combined with GM-CSF and IL-3 or CKL, TPO can generate cells of the megakaryocytic lineage and maintain or increase the number of HPCs in our suspension culture system. For pretransplant expansion, TPO combined with GM-CSF and CKL is a promising combination.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation of Bone Marrow CD34+ Cells
One hundred ml of heparinized bone marrow were aspirated from the posterior iliac crest of healthy, consenting adults. The samples were spun twice for 10 min at 200 g to remove an excess of 90% of platelets, including platelet aggregates. Mononuclear cells (MNCs) were prepared by density gradient centrifugation (1.077 g/ml, Lymphoprep; Nycomed Pharma; Oslo, Norway). CD34+ cells were immunomagnetically isolated as described elsewhere [21]; enumerated and phenotyped by flow cytometry.

Cultures and Growth Factors
Isolated CD34+ cells were cultured in medium in 96-well tissue culture plates at 37° C in humidified air with 5% CO2, applying 10,000 cells in a final volume of 200 µl Iscove's modified Dulbecco's medium (IMDM) (Bio Whittaker; Verviers, Belgium) supplemented with 10% fetal bovine serum (GIBCO; Paisley, Scotland), 2 mmol/l glutamine (GIBCO), 100 µg/ml streptomycin (GIBCO), and 60 µg/ml penicillin (Apothekernes Laboratorium; Oslo, Norway) per well. The following GFs were added in all possible combinations: 100 IU/ml TPO (a kind gift from ZymoGenetics; Seattle, WA), 2 IU/ml CKL, 80 IU/ml IL-3, and 80 IU/ml GM-CSF (all kindly provided by Genetics Institute; Cambridge, MA). Culture medium without GFs served as negative control. The culture media were supplemented as needed, with medium and GFs added in appropriate concentrations. After 7, 10, and 14 days, the cells were harvested; viable cells were enumerated using acridine orange, ethidium bromide staining, and fluorescence microscopy; and the cells were immunophenotyped by flow cytometry analyses. Six experiments were initially run. Additionally, four experiments using the five GF combinations that expanded megakaryocyte precursors and HPCs most efficiently in the initial experiments were also performed.

Flow Cytometry and Analyses
Cell surface molecule expression was determined on CD34+ cells after isolation and on harvested cells after 7 and 14 days in culture. We double-stained with fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-labeled anti-CD34 (anti-HPCA-2) monoclonal antibodies (mAbs), anti-CD61 (FITC) and anti-CD42a (FITC), anti-HLA-DR (FITC), anti-CD38 (PE), anti-CD19 (FITC), and anti-CD33 (FITC). Simultest control (FITC IgG1 + PE IgG2a) served as a negative control. All mAbs were purchased from Becton Dickinson Immunocytometry Systems; San Jose, CA). Acquisition and analysis were performed on a FACSort (Becton Dickinson) flow cytometer equipped with an air-cooled argon-ion laser tuned at 488 nm. Data were collected in list mode files using CellQuest (Becton Dickinson) for acquisition. The CellQuest and Attractors (Becton Dickinson) software packages were used for analyses. In the Attractors software, we developed attractors that could be applied in all analyses for megakaryocyte lineage-associated molecules and CD34. Statistical analyses utilized t-test on pooled results of all combinations with and without TPO. Statistical significance was set to p < 0.05.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Phenotype of CD34+ HPCs
CD34+ bone marrow HPCs with a purity of 97.8% (mean; range 95.6%-99.1%, standard deviation = 1.4%). Table 1 shows the expression of the megakaryocyte lineage-associated cell surface molecules CD61 and CD42a on CD34+ HPCs. As can be seen, the relative number of CD34+ cells expressing CD61 and CD42a was 2.2% and 9.2%, respectively. Thus, a small but significant population of CD34+ bone marrow HPCs expressed megakaryocyte lineage-associated cell surface molecules. However, due to large interpersonal variations, the standard deviations were as high as 1.1% and 4.6%, respectively.


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  		Table 1. Relative distribution of megakaryocyte lineage-associated cell surface molecules on CD34+ bone marrow HPC. Analysis performed on cell immediately after marrow aspiration, i.e., day 0.
 
Effect of Cultivating CD34+ HPCs in the Presence of TPO
TPO had a profound effect on the cultured CD34+ cells. This is demonstrated in Figure 1, which illustrates the differences in phenotype of cells cultured for 14 days in the presence of GM-CSF and CKL, with or without TPO present. The percentage of each subpopulation is given in the plot quadrants and shows that more cells expressing CD34+/–CD61+, CD34+CD61+/–, and CD34+CD61+ were generated in the culture where TPO was present. When pooling the data from all GF combinations ( Fig. 2), the presence of TPO during 14 days of culture yielded a significantly increased number of cells expressing the megakaryocytic lineage-associated cell surface molecule CD61 (p < 0.01). We also found a significant increase in the number of cells of the megakaryocytic lineage with a more mature phenotype CD61+CD34– (p < 0.01), as well as of cells with the nonmegakaryocytic phenotype CD61–CD34+ (p < 0.01), i.e., progenitor cell phenotype. The number of CD34+ cells and the total number of cells were also significantly increased, p < 0.01 and p = 0.05, respectively, when TPO was present in the cultures. The content of the megakaryocytic precursors CD61+CD34+ was, however, not statistically affected by addition of TPO.



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  		Figure 1. Dot plot from a typical experiment showing two GF combinations, in this case GM-CSF and CKL, with TPO (right plot), and without TPO (left plot). The CD34+ (vertical axis), the CD61+ (horizontal axis), and the CD61+ CD34+ (upper right quadrant) populations are larger in the culture containing TPO. Both plots are gated in forward and side scatter on viable cells, showing a total of 42,000 (without TPO), and 44,000 (with TPO) events; total numbers of events analyzed were 50,000 for both analyses.

 


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  		Figure 2. Relative average increases of subsets on day 14 in cultures containing TPO in the top row and without TPO in the bottom row of each GF combination. Scores are sorted after relative change of CD61+ cells (A). The Y-axis gives amplification factor where the digit 1 indicates no increase or decrease.

 
Furthermore, the individual GF combinations with TPO present or absent were pooled and compared by analyzing the stimulation indices, i.e., dividing the number of a given cell population generated after 14 days in culture to the correlating number of cells in the corresponding CD34+ subset at day 0. This is demonstrated in Figure 2, which shows that CD61+ cells increased as much as 119 times ( Fig. 2A) and the CD61+CD34– cells 302 times ( Fig. 2B) with the most efficient GF combination (TPO + GM-CSF + IL-3). The combination of GM-CSF and IL-3 without TPO, on the other hand, caused only 39-fold and 80-fold expansions of the CD61+ and CD61+CD34– cells, respectively. As can be seen in the figure, higher expansion of the two subsets in the presence of TPO is an evident trend, although not statistically significant in the present material, probably due to the low number of experiments and high interpersonal variations. Further, TPO also gave a significant increase in CD34+ cells for most GF combinations ( Fig. 2C), but the TPO-driven expansion was much more extensive for the CD61+CD34+ subpopulation ( Fig. 2D); e.g., TPO + GM-CSF increased the number of CD61+CD34+ cells 50 times. For CD61–CD34+ cells ( Fig. 2E) and total cell yield ( Fig. 2F), we also found an apparent increase when adding TPO.

The GF combinations with TPO and GM-CSF present gave the highest yield of megakaryocytic progenitors, megakaryocytic lineage cells, and HPCs. The addition of IL-3 or CKL to TPO and GM-CSF added to this effect. The addition of IL-3 caused the highest increase in megakaryocytic cells ( Figs. 2A and 2B), while CKL addition generated more HPCs ( Figs. 2C and 2E), at the same time yielding the second largest number of megakaryocytic cells. The full four-GF combination was on average the most efficient cell and CD34+ expander, but fewer cells of the megakaryocytic lineage were generated.

Kinetics of Cultivated CD34+ Cells
When looking at the expansion of cell subsets at days 7 and 14 in cultures containing TPO and GM-CSF combined with IL-3 or CKL ( Fig. 3), both TPO and GM-CSF combined with IL-3 or CKL initially stimulated expansion of CD61+ and CD61+CD34– cells as well as total cell number. During the second week of cultivation, the presence of CKL increased expansion of all cell subsets while the presence of IL-3, on the other hand, caused an increase, decrease, or no effect on the number of cells. Thus, the presence of CKL together with TPO and GM-CSF adds invariably to the expansion of HPCs and megakaryocytic cells.



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  		Figure 3. Data from one bone marrow donor CD34+ HPC at days 7 and 14. Addition of TPO (shaded circles) is consistent with higher yields in all subsets when compared with the same GF combination without TPO added (open circles). The X-axis gives amplification factor where the digit 1 indicates no increase or decrease.

 
Generation of Other Cell Subsets
For the two most interesting GF combinations, i.e., TPO and GM-CSF in combination with IL-3 or CKL, we also analyzed the cultured cells for their expression of CD34+HLA-DR+, CD33+ and CD38, as summarized in Table 2. TPO in combination with IL-3 and GM-CSF generated higher proportions of the CD38dullCD34+ subset than TPO, CKL, and GM-CSF. This difference is not statistically different but shows a trend towards the generation of more CD38dullCD34+ in the presence of TPO, IL-3, and GM-CSF. On the other hand, TPO, CKL, and GM-CSF generated higher proportions of the HLA-DR+, HLA-DRdullCD34+, and CD33+ subsets.


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  		Table 2. Comparison of cell phenotypes in absolute number of cells after 14 days in culture, starting with 10,000 CD34+ cells at day 0. Two SD in parentheses.
 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Some of the most serious complications of stem cell transplantation are derived from neutropenia and thrombocytopenia. Adding HPCs that have been expanded ex vivo to the graft is thought to be one promising way of reducing these complications. Culturing HPCs in an optimal mixture of GFs can selectively enrich for given cell subsets. To study what effect TPO might have on HPC expansion and to find an efficient combination of GFs for ex vivo expansion of cells of the megakaryocytic lineage and of HPCs, we cultured HPCs for 14 days in the presence of various GF combinations. Our results show that TPO significantly participates in increasing the number of megakaryocytic cells and HPCs and that the content of megakaryocytic progenitors is also maintained or increased in optimal GF combinations.

Table 3 compiles our findings by showing relative change in cell numbers of megakaryocyte lineage-associated cells and HPC subsets after 14 days of culture. As can be seen, there is an evident trend of higher increases in cell numbers in cultures containing TPO than in the cultures without TPO present.


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  		Table 3. Relative changes in cell numbers*
 
There are several other interesting GFs which are known as early-acting and beneficial for expansion of HPCs along the megakaryocytic lineage, among the most interesting being IL-6, IL-11, macrophage inflammatory protein-1{alpha}, and FLT3. To keep the experimental set-up manageable and to avoid mobilization of all HPCs in future clinical applications, we chose to omit these GFs. Mature cells would be of no use to a patient in the most critical pancytopenic period, and the finding that IL-6 addition to cultures yields more mature megakaryocytic lineage cells than CKL [22] prompted us to exclude IL-6. We are, however, expanding this study by comparing the effect of adding FLT3 to TPO, GM-CSF, and CKL to see if this might further enhance the graft and maintain or increase the content of HPCs. Studies combining megakaryocyte growth and development factor (MGDF), erythropoietin, FLT3, G-CSF, stem cell factor, IL-3, and IL-6 have shown a great potential for expanding HPCs, although not towards increased generation of CFU-Mks. The presence of FLT3 and TPO were especially effective towards the generation of long-term culture initiating cells [23].

Looking at all GF combinations, TPO gives a significant increase in the CD61+ cell subset, and the best GF combinations for expanding the CD61+ megakaryocytic cell populations are TPO, GM-CSF, and IL-3; TPO, GM-CSF, and CKL; and TPO and IL-3. The same GF combinations give an even higher increase in the number of more mature megakaryocytic subset, i.e., CD61+CD34– cells. Finally, the expansion of megakaryocytic progenitor cell populations, i.e., the CD61+CD34+ subset, was most pronounced with TPO combined with GM-CSF and IL-3 or with GM-CSF alone. The CD42a data correlate closely with the CD61 data (results not shown). Taken together, the combination of TPO with GM-CSF and CKL gives the best outcome for the expansion of cell populations of the megakaryocytic lineage including megakaryocytic progenitor cell subsets. This result is concordant with previous studies of TPO in in vitro cultures [10, 16] where the megakaryocytopoietic effect is enhanced with the addition of IL-3 or CKL [15, 24, 25] and with a study of the effect of IL-3 on megakaryocytic development [26].

Another effect of TPO is the expansion of the CD34+ HPC subset. There are almost twice as many cells expressing CD34 in the GF combinations of CKL, GM-CSF, IL-3, and TPO; CKL, GM-CSF, and TPO; and CKL, IL-3, and TPO, after 14 days in culture as compared with the number of cells incubated. Although the differences between the individual experiments make the effect of TPO difficult to assess statistically for the individual GF combinations, there is an overall significant increase in CD34+ cell numbers during this cultivation. Numerically, the effect is most pronounced with TPO combined with GM-CSF and CKL. Similar findings in a murine system [14] support our results. Thus, looking at the results as a whole, the most efficient GF combination to expand cell populations of the megakaryocytic lineage including megakaryocytic progenitors as well as CD34+ cells and CD61–CD34+ cells is TPO combined with GM-CSF and CKL. This GF combination is both a very effective expander of cell subsets of the megakaryocytic lineage, which might be of importance in decreasing the thrombocytopenia after stem cell transplantation, and of HPC subsets, which could accelerate engraftment. This conclusion also seems to hold for serum-free cultures that we currently study for applying expanded cells to preclinical transplantation. However, with no serum present, we have a significantly lower cell yield, but this is partly compensated by an increase in the yield of CD61+ cells. Further studies will be performed to optimize the conditions for ex vivo expansion in the presence of serum-free culture media.

The most effective GF combinations contained TPO and GM-CSF. The addition of IL-3 further expands cell subsets along the megakaryocytic lineage. Evidently, TPO, GM-CSF, and IL-3 is the best GF combination for the ex vivo expansion of the megakaryocytic lineage. The second-best GF combination, TPO, GM-CSF, and CKL, simultaneously gives better maintenance or expansion of megakaryocytic cell and progenitor cell populations and the CD34+ HPC subset. The addition of both CKL and IL-3 gives the best overall yield while at the same time driving the cells further towards maturity. Based on these interpretations, we have decided to apply TPO, GM-CSF, and CKL in a preclinical transplantation setting for ex vivo stem cell expansion with transplantation of marked expanded cells in a monkey system for further characterization of the effect of our expansion protocol.

We have based our culture system on 14 days' expansion. Most pretransplant expansion models last for only seven days. Based on the dynamics and the high endogenous levels of TPO in the myeloablated patients [9], it is possible that by applying our GF combination, sufficient numbers of progenitors will be expanded and activated to reduce thrombocytopenia and accelerate engraftment after transplantation. We are currently planning to add cells from seven days' expansion to unexpanded stem cells in an animal transplantation model.

TPO in itself has been shown to be a powerful megakaryocyte population expander in vivo and in vitro, and it has earlier been shown to act synergistically with other hematopoietic GFs both in murine and human models [14-17]. This is in accordance with our findings. In a clinical model using MGDF, i.e., a truncated molecule related to TPO, and several other cytokines for expansion of bone marrow grafts [27], optimal expansion time for megakaryocyte lineage-associated cells was found to be seven days with a 58-fold expansion of CD61+ cells. Using only the three GFs—TPO, GM-CSF, and CKL—we found that expansion continued beyond seven days to reach higher final yield, and our dynamic study gives good hope for additional expansion in vivo beyond the first week. This study and the study on in vivo TPO administration to myeloablated monkeys showing a reduced need for platelet transfusion without any signs of serious side effects [28] make transplantations of cell populations expanded with TPO combined with other GFs worth evaluating in preclinical trials.

To conclude, the results of our study indicate that in a future transplantation setting it might be feasible to expand a human bone marrow stem cell graft ex vivo in easy-to-manage culture systems with the addition of TPO, GM-CSF, and CKL, the result being a graft with the potential of decreased need for platelet transfusions and possibly a reduction of other complications caused by the pancytopenia after myeloablative treatment.


   Acknowledgments
 
We thank Lill Anny Gunnes Grøseth for excellent technical assistance and ZymoGenetics and Genetics Institute for supplying growth factors.

This work was supported by a Contract of the Commission of the European Communities.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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accepted for publication on November 5, 1998.




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STEM CELLS THE ONCOLOGIST CME ALPHAMED PRESS JOURNALS