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Stem Cells, Vol. 18, No. 6, 428-434, November 2000
© 2000 AlphaMed Press

In Vitro Proliferation and Differentiation of Megakaryocytic Progenitors in Patients with Aplastic Anemia, Paroxysmal Nocturnal Hemoglobinuria, and the Myelodysplastic Syndromes

Charlotte V. Cox, Sally B. Killick, Sandeep Patel, Moldupe O. Elebute, Judith C.W. Marsh, Edward C. Gordon-Smith, Frances M. Gibson

Department of Hematology, St. George's Hospital Medical School, London, UK

Key Words. Megakaryocytopoiesis • Aplastic anemia • Paroxysmal nocturnal hemoglobinuria • Myelodysplastic syndromes

Frances Gibson, Ph.D., Dept of Haematology, St George's Hospital Medical School, Cranmer Terrace, London SW17 ORE, United Kingdom. Telephone: 0208-725-5481; Fax: 0208-725-0245; e-mail: fgibson{at}sghms.ac.uk


   ABSTRACT
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It has previously been shown that patients with aplastic anemia (AA) have a stem cell defect both of proliferation and differentiation. This has been shown by long-term bone marrow (BM) culture, long-term initiating cell assays, and committed progenitor assays. We present, for the first time, data on megakaryocyte (Mk) colony formation from purified BM CD34+ cells from patients with AA. The results are compared with those from normal controls and from patients with paroxysmal nocturnal hemoglobinuria (PNH) and the myelodysplastic syndromes (MDSs). Those treated for AA had previously received immunosuppression (antithymocyte globulin and/or cyclosporin). No patients had received bone marrow transplantation. A total of 13 AA patients (five untreated, eight treated), six PNH, six MDS, and 13 normal donors were studied. BM CD34+ cells were purified by indirect labeling and then cultured in a collagen-based Mk assay kit (MegaCult-C, StemCell Technologies). The cultures were fixed on day 12, and the Mk colonies were identified by the alkaline phosphatase anti-alkaline phosphatase technique using the monoclonal antibody CD41 (GP IIb/IIIa). The slides were scored for Mk colony-forming units (CFU-Mks) (3-20 and >20 cells), Mk burst-forming units (BFU-Mks) (>50 cells), and mixed colonies.

The results show that total Mk colony formation in AA was significantly lower than in normal donors (p < 0.0001), both in untreated patients/nonresponders to treatment (p = 0.0001) and in complete/partial responders (p < 0.002). There was no significant difference in Mk colony formation in treated and untreated patients (p = 0.05). Patients with AA had a lower total colony formation than PNH patients (p = 0.0002). PNH patients exhibited lower colony formation than normal controls (p = 0.03), as shown by MDS patients, although the considerable number of variables resulted in a lack of statistically significant difference from normal controls (p = 0.2).

We have now shown that Mk colony formation from purified BM CD34+ cells is significantly reduced, supporting previous evidence that AA results from a stem cell defect.


   INTRODUCTION
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Previous studies have demonstrated that patients with aplastic anemia (AA) have a reduction or total absence of bone marrow progenitor cells in all cell lineages, specifically granulocyte-monocyte colony-forming units (CFU-GMs) and BFU-Es, as well as progenitor cell production in long-term bone marrow culture (LTBMC) [1-3]. AA bone marow (BM) also shows a deficiency in the proportion of CD34+ [4] and earlier CD34+/CD33– cells [5], indicating a defect at the level of the hemopoietic stem cell. Crossover studies using CD34+ cells cultured onto normal stroma again show the absence of progenitor cell production, suggesting a deficiency of long-term culture initiating cells [6], which have been quantified in several studies and shown to be reduced [7-9]. To date, however, there has only been one published paper describing megakaryocytopoiesis in AA [10] from unpurified BM cells which showed significantly reduced megakaryocyte colony-forming units (CFU-Mks) compared with levels in normal donors. In patients with paroxysmal nocturnal hemoglobinuria (PNH), in vitro culture studies have shown a marked decrease in erythroid and myeloid progenitor cells in peripheral blood and BM [11-16], but there are very few data on megakaryocytopoiesis in PNH [12]. There is general agreement that patients with myelodysplastic syndromes (MDS) have pronounced deficiencies of both colony formation of CFU-GMs, BFU-Es, and CFU-Mks in clonogenic culture and from LTBMCs in a significant proportion of patients [17-23]. The majority of patients (55%-65%) have defective colony formation in all three cell lineages [18, 21] and the deficiency of growth increases with disease severity and French-American-British (FAB) score [21-23].

For the first time, we present the results of Mk colony formation from purified CD34+ BM cells, thus eliminating contaminating accessory cells. Mk colony formation from patients with AA, PNH, and MDS was compared with that in normal donors.


   MATERIALS AND METHODS
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mk colony growth was assessed in 13 patients with acquired AA (five untreated, eight treated), six patients with hemolytic PNH, six patients with MDS (three refractory anemia, one refractory anemia with ringed sideroblasts, one refractory anemia with excess blasts, and one refractory anemia with excess blasts in transformation) and 13 normal controls. Patient details and previous treatment are shown in Table 1Go. Diagnosis and severity of disease for the AA patients were classified according to peripheral blood and BM findings [24, 25] and for the MDS patients according to published guidelines [26, 27]. All AA patients had a negative Ham's test and normal expression of glycosylphosphatidylinositol (GPI)-anchored proteins on their peripheral blood cells. The diagnosis of PNH was made by a positive Ham's test and/or demonstration of a deficiency of GPI-anchored proteins on their peripheral blood cells. BM was obtained from normal donors after informed consent, in keeping with local hospital ethical committee guidelines.


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  		Table 1. Patient characteristics
 
Preparation of Bone Marrow Mononuclear Cells (BMMCs)
Bone marrow was collected in 10 IU/ml preservative-free heparin (Leo; Princes Risborough; Bucks, UK) from posterior iliac crest marrow aspirates. Marrow was diluted 1:1 in Iscove's modified Dulbecco's medium (GIBCO; Paisley, Scotland), supplemented with 10% fetal calf serum (FCS), 100 IU/ml penicillin-streptomycin (GIBCO), and 100 IU/ml L-glutamine. The marrow was centrifuged on Ficoll-hypaque at 400 g for 30 min at room temperature to obtain BMMCs, which were then washed twice. Cell number and viability were assessed after trypan blue staining.

Purification of CD34+ Cells
CD34+ cells were isolated from BMMCs by indirect magnetic labeling (MACS; Miltenyi Biotec; Bisley, Surrey, UK; http://www.miltenyibiotec.com). BMMCs were washed twice in buffer consisting of phosphate-buffered saline supplemented with 0.5% bovine serum albumin (BSA), 0.6% acid citrate dextrose, and 4% sodium bicarbonate. Cells were incubated with i/human {gamma}-globulin (100 µl/108 BMMCs) and monoclonal hapten-conjugated anti-CD34 antibody (QBEND/10) (100 µl/108 BMMCs) for 15 min at 6-12°C. The cells were washed in buffer followed by incubation with ii/ colloidal super-paramagnetic MACS Microbeads conjugated to an anti-hapten antibody (100 µl/108 BMMCs) for 15 min at 6-12°C. The cells were washed and resuspended in 500 µl buffer per 108 BMMCs. The magnetically labeled cells were enriched on a positive selection column in the magnetic field of the MiniMACS. Cell count and viability were assessed after separation using trypan blue.

MegaCult CFU-Mk Assay
A commercial Mk assay detection kit (MegaCult-C, StemCell Technologies, Inc.; Vancouver, Canada; http://www.stemcell.com) was used to quantitate Mk colonies. Viable CD34+ cells were added at 2.5 x 104 cells per chamber (in 0.75 ml of medium) of a double-chamber slide. This collagen-based system contained a medium supplemented to a final concentration with 1.1 mg/ml collagen, 1% BSA, 0.01 mg/ml bovine pancreatic insulin, 0.2 mg/ml human transferrin (iron saturated) and the human recombinant cytokines: 50 ng/ml thrombopoietin (TPO), 10 ng/ml interleukin 3 (IL-3), and 10 ng/ml IL-6. The chamber slides were incubated at 37°C for 10-14 days and then fixed for 20 min in 1:3 methanol/acetone.

Immunocytochemical Analysis of CFU-Mks
The Mk colonies were immunocytochemically examined using a monoclonal anti-CD41 (GPIIb/IIIa) (Dako; Ely, Cambs, UK; http://www.dako.dk) by APAAP (alkaline phosphatase anti-alkaline phosphatase) technique. Briefly, the chamber slides provided in the kit contained two square areas; each was scored with a circle, a quarter of the square area. Slides were allowed to stand at room temperature for 30 min and then fixed for 90 sec. Human {gamma}-globulin was added for 10 min. The slides were washed between each step with 0.05 M Tris/NaCl buffer pH 7.6 (TBS). The primary antibody mouse anti-human GPIIb/IIIa (CD41) (Dako) was diluted 1:5 in TBS and added for 30 min. Rabbit anti-mouse immunoglobulin (1:10 TBS) with 4% normal human serum and APAAP (1:10 TBS) were added for 30 min and then these incubations were repeated for 10 min each. Substrate solution was prepared with 100 mg N XS phosphate, 200 µl amide, 9.8 ml buffer pH 8.5, 60 µl levamisole (1.024 g/5 ml, Sigma; Poole, Dorset, UK; http://www.sigma-aldrich.com) and 200 mg fast red salt (Sigma). This was filtered and then flooded over the slides for 20 min. Slides were washed with distilled water and counter-stained with hematoxylin (mercury-free; BDH Ltd., Poole, Dorset, UK) for 10 min. Slides were immersed in water, washed for 5 min and then left to dry.

Scoring Procedures
Mk colonies were microscopically identified and scored. Three categories of colonies were identified: pure Mk colonies, mixed Mk colonies (distinguished by the presence of non-Mk cells within the same colony; Fig. 1AGo), and non-Mk colonies. Pure Mk colonies were scored according to size and maturity: CFU-Mks of 3-20 cells (Fig. 1BGo), CFU-Mks of >20 cells representing colonies from mature precursor cells and megakaryocyte burst-forming units (BFU-Mks) where the colonies had >50 cells spreading from a central area (Fig. 1CGo), representing colonies from primitive precursor cells. Results were also expressed as total colonies, which included pure and mixed Mk colonies.





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  		Figure 1. Megakaryocyte colonies from bone marrow CD34+ cells; A) mixed Mk colony; B) CFU-Mks 3-20 cells; C) BFU-Mks.

 

   RESULTS
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The purity of CD34+ cells was analyzed when sufficient cells were available. Mean purity in normal donors was 90% (84%-98%), in PNH 90% (78%-99%), and in MDS 97% (96%-98%). Cell numbers were too low in AA to permit analysis.

The results of Mk colony formation from normal, AA, hemolytic PNH, and MDS bone marrow CD34+ cells are shown in Figures 2A-2DGo. Total Mk colony formation (per 2.5 x 104 CD34+ cells) in AA (mean 11 ± 20, range 0-64) was lower than in normal donors (mean 107 ± 53, range 16-180) (p < 0.0001), both in untreated patients (mean 3 ± 2, range 0-8) or nonresponders to treatment (mean 0) (p = 0.0001) and in complete (mean 54 ± 14, range 44-64) or partial responders (mean 7 ± 5, range 0-12) (p < 0.002). Mk colony formation in complete responders was significantly higher than in both patients who had not responded (p < 0.05), and those with partial response (p < 0.005).




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  		Figure 2. Megakaryocyte colonies from bone marrow CD34+ cells in A) normal donors; B) aplastic anemia; C) paroxysmal nocturnal hemoglobinuria, and D) myelodysplasia.

 
Patients with AA had lower Mk colony formation than PNH patients (mean 54 ± 15, range 40-72) (p = 0.0002). PNH patients had lower Mk colony formation than normal donors (p < 0.03). In MDS patients, Mk colony numbers (mean 64 ± 74, range 0-156) were reduced to numbers comparable to those in PNH patients, although due to the considerable variation showed no significant difference from normal donors.

MK colony numbers did not correlate with platelet counts in any patient group.


   DISCUSSION
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Megakaryocytopoiesis is a complex series of events [28] which has been studied using a variety of in vitro clonal assays. Cells of the Mk lineage are susceptible to inhibitors such as transforming growth factor-ß (TGF-ß), often present in FCS [29], which has previously caused difficulties in growing Mk colonies [30]. The availability of recombinant cytokines, most importantly IL-3 [30], IL-6 [31], IL-1 [32], TPO [33, 34], and stem cell factor [35], has allowed the development of serum-free cultures for routine quantitation of Mk colonies. A variety of semisolid matrix materials have been used to support the growth of Mk progenitors that allow specific immunocytochemical staining to be applied to the whole culture. Serum-free fibrin clots [30], agar [36], and collagen have all been used to quantitate colonies of the Mk lineage. The MegaCult-C assay has been developed and validated to allow detection and quantitation of CFU-Mks from BMMCs and CD34+ cells in a collagen-based system which has growth factors added in a concentration optimal for Mk growth [37, 38]. We found the assay to be quick and simple, and it elevated Mk colony definition to a high standard. Together with a strict scoring system, we have been able to incorporate CFU-Mk quantitation into our cell culture laboratory, having established our own normal values.

In this study, we present data on Mk colony formation from purified BM CD34+ cells from patients with AA, hemolytic PNH, and MDS. Previous studies [10, 17, 20, 22] have used unpurified BM cells, which makes analysis of the results more difficult, since contaminating accessory cells release a variety of hemopoietic growth factors and confound the interpretation of results. As with all other clonogenic assays there was considerable interindividual variation, even among the normal donors. We have shown that all AA patients have reduced early (BFU-Mk) and late (CFU-Mk) Mk colony formation which does not significantly improve in those patients who have achieved a response from immunosuppressive therapy (including a platelet response). Previous studies have similarly shown poor growth of CFU-GMs, CFU-GEMs (composed of granulocyte, erythroid, and monocyte cells), and BFU-Es in AA patients responding to immunosuppressive therapy [3, 39, 40]. In keeping with previous in vitro culture studies in PNH showing reduced colony formation (CFU-GMs and BFU-Es) [11-13, 15, 16], Mk colony formation was lower than that in normal donors but significantly higher than colony formation in AA patients. In patients with MDS, Mk colony formation was lower than normal, although highly variable. We did not find that colony formation was related to disease progression or transfusion dependence, although the low patient numbers within each FAB subtype do not allow valid conclusions.

We have quantitated another hemopoietic progenitor cell in AA and shown that Mk colony formation from purified CD34+ cells is significantly reduced, supporting previous evidence of a stem cell defect in AA; it remains to be determined whether the cause of this defect lies within or outside the stem cell.


   ACKNOWLEDGMENTS
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This work was supported by Amgen, IMTIX Sangstat, The Marrow Environment Fund, and StemCell Technologies, Inc.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received June 28, 2000; accepted for publication August 30, 2000.




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