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Stem Cells, Vol. 14, No. 5, 577-583, September 1996
© 1996 AlphaMed Press

ORIGINAL PAPER

Blast Colony Forming Cell-Binding Capacity of Bone Marrow Stroma from Myelodysplastic Patients

Julia Gidáli, I. Fehér, Susan R. Hollán

National Institute of Hematology and Immunology, Budapest, Hungary

Key Words. Stroma-adherent blast colony-forming cells (CFU-BL) • Bone marrow stroma • MDS • Hemopoietic growth factors

Correspondence: Dr. J. Gidáli, National Institute of Hematology and Immunology, Budapest, PO Box 44, H-1518, Budapest, Hungary.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
A specific stroma function can be quantitatively assessed by counting the stroma-adherent blast cell colonies (CFU-BL) that are formed from normal plastic nonadherent mononuclear bone marrow cells (PNAMNC) after a short-term coincubation ("panning") with the preformed stromal layer. In order to obtain information of stroma function in myelodysplasia (MDS), the "CFU-BL-binding capacity" of stroma from normal bone marrow and from patients with MDS were compared. Stromal cell cultures were established from mononuclear bone marrow cells in microplate cultures cultured with or without 10–6 M hydrocortisone. CFU-BL-binding capacity was studied by counting blast colonies seven days after panning, and the results were expressed as CFU-BL/103 PNAMNC. Normal marrow stromal layers bound CFU-BL only if they were cultured with hydrocortisone, while MDS stromal layers also bound CFU-BL in the absence of hydrocortisone. For further studies of the function of MDS stroma, the effect of growth factors (stem cell factor [SCF], G-CSF, interleukin 3 [IL-3] and their combinations) on CFU-BL binding by normal or MDS stroma has also been compared. Twenty-hour incubation of the stromal layers with a standard dose (100 ng/ml) of various hemopoietic growth factors (IL-3 alone or in combination with SCF, G-CSF alone or in combination with SCF) did not have any effect on CFU-BL binding by normal marrow stroma, but increased the CFU-BL binding by stromal layers from MDS bone marrow. These findings suggest that although stromal microenvironment in MDS is capable of supporting hemopoiesis, bone marrow stroma from MDS patients differs in some characteristics from the normal stroma.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Myelodysplastic syndrome (MDS) is a clonal disorder of the hemopoietic stem cell characterized by a spectrum of clinical and morphological changes in hemopoiesis. Cytopenia is associated with morphological signs of maturation inhibition [1]. Quantitative and qualitative defects of progenitors are frequent findings [2-5]. A recent review stressed that in spite of ample literature on in vitro bone marrow studies, no consistent diagnostic abnormalities have been detected so far [6].

The role of the stroma in the regulation of normal hemopoiesis is widely accepted. The fact that acute nonlymphocytic leukemia occurs in a high percentage of MDS patients renders MDS a useful model for studying the behavior of the hemopoietic microenvironment in connection with leukemic transformation.

The effects of various cytokines on the progenitors in MDS patients have been studied thoroughly [7, 8] and even some clinical implications were offered [9]. Relatively few data are available, however, on the function of stromal cells, as well as the role of regulatory molecules in the function of the hemopoietic microenvironment in MDS patients [10, 11]. In a recent paper, an adherent layer from normal MDS and acute myeloid leukemia (AML) bone marrows were found to be able to produce elevated cytokine levels in a significant percentage of patients [12].

The hemopoietic microenvironment is a complex entity. Quantitative evaluation of its function needs special techniques. One of these is the assay of the so-called fibroblast colony forming cell (CFU-F) [13] which enumerates one of the components of the complex microenvironment. Another technique qualitatively characterizes the hemopoiesis-supporting function of the microenvironment by counting a primitive progenitor cell type from normal bone marrow which does not adhere to plastic, but which adheres to preformed human marrow derived from stromal layers and forms colonies of blast cells, blast colony forming cells (CFU-BL) [14-16]. In the present paper, this "blast cell-binding capacity" of bone marrow stromal layers from normal control and from patients with MDS was compared to characterize the stromal function of MDS patients. For further studies of the function of MDS stroma, the effect of growth factors (stem cell factor [SCF], G-CSF, interleukin 3 [IL-3] and their combinations) on the CFU-BL binding by normal or MDS stroma has also been compared.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Stromal Layers
Bone marrow samples from MDS patients were obtained from sternal puncture. Control samples were taken from normal donors for allogeneic bone marrow transplantation (after having received their informed consent). Mononuclear bone marrow cells were separated by density gradient centrifugation (Percoll; Pharmacia, Sweden, 1.077 g/cm3).

Stromal layers were cultured by the technique of Gordon et al. [14] and modified in our laboratory [17]. Briefly, 1 x 104 mononuclear bone marrow cells/100 µl were placed in a flat bottom 96-well tissue culture plate (Greiner; Nürtingen, Germany). Tissue culture medium consisted of {alpha}-minimal essential medium (MEM) (GIBCO Europe) supplemented with 15% fetal calf serum (FCS) (SEBAK; Germany). Cultures were grown in the presence or absence of 10–6 M hydrocortisone hemisuccinate (Sigma-Aldrich; Milwaukee, WI). Cultures were maintained at 37°C in 5% CO2-air. Medium was changed completely at weekly intervals. Time for receiving a confluent stromal layer was usually four weeks for normal bone marrow, while MDS stromal layers usually reached confluence one week earlier. Cultures were used when hemopoiesis (round cells on the surface) ceased to be observed.

Panning of Bone Marrow Cells (CFU-BL Assay)
Adherent cells were removed from mononuclear fractions of normal bone marrow by incubation in plastic tissue culture flasks for 2 h. After having removed the supernatant, plastic nonadherent mononuclear bone marrow cells (PNAMNC) were added to the established stromal layer at a concentration of 1 x 103/well. After 2 h coincubation, the supernatant was removed and the stromal layer was carefully washed three times to remove nonattached cells. Then the stromal layer was covered with 100 µl of 0.3% agar in {alpha}-MEM + FCS. A minimum of 6, and usually 12 parallel wells were used. Colonies were counted by inverted microscope on day 7 and colony count was expressed as CFU-BL/103 PNAMNC. Using established stromal layers from normal bone marrows cultured in the presence of hydrocortisone, a strict linear correlation between panned PNAMNC and colony formation was observed. Morphology of the colonies corresponded to that described by Gordon et al. containing blasts, immature and mature granulocytic cells [14]. A typical colony is shown in Figure 1Go.



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  		Figure 1. Blast cell colony grown on normal bone marrow stroma cultured with hydrocortisone seven days after panning. Original magnification 200x(phase contrast illumination).

 
Cytokines
Cytokines (G-CSF, IL-3 and SCF; Amersham International; Amersham, United Kingdom) were added to the stromal cultures 20 h before panning the PNAMNCs. If not otherwise stated, cytokines were added in a standard dose of 10 ng/well.

Statistical Analysis
Linear regression and t-test were calculated by Statgraf computer program.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
As it has been stated in previous reports [18], normal CFU-BL adhered to and formed colonies on normal stroma cultured with hydrocortisone (HC) but not on HC-noncontaining normal stroma. On marrow stroma derived from nine MDS patients, however, normal CFU-BL adhered to and formed colonies on stroma cultured without HC, too (Table 1Go).


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  		Table 1. Blast cell-binding capacity of stromal layers from normal and MDS bone marrow grown in the presence or absence of 10–6 M hydrocortisone
 
Short-term incubation (20 h) with a standard dose (10 ng/well = 100 ng/ml) of various hemopoietic growth factors (IL-3 alone or in combination with SCF, G-CSF alone or in combination with SCF) did not have any effect on adherent CFU-BL binding to normal marrow stroma. Neither stromal layers cultured without HC nor those with HC showed increased CFU-BL binding (Table 2Go).


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  		Table 2. The effect of various growth factors on the blast cell-binding capacity of stromal layers from normal bone marrow grown in the presence or absence of 10–6 M hydrocortisone
 
Under the same conditions, however, these growth factors (IL-3 alone or combined with SCF, G-CSF alone or combined with SCF), increased the colony formation from normal PNAMNC on stromal layers from MDS bone marrows cultured with HC (Table 3Go) or without HC (Table 4Go). This effect was exerted by all stromas obtained from patients belonging to different subtypes of MDS but, due to the wide variation, not each experiment showed a statistically significant difference. Refractory anemia with excess of blasts (RAEB) stroma grown with or without HC supported the adherence and colony formation of normal CFU-BL less than refractory anemia (RA) or refractory anemia with ringed sideroblasts (RAS). Due to the low number of individual experiments, this difference was not statistically significant.


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  		Table 3. The effect of various growth factors on the blast cell-binding capacity of stromal layers from MDS bone marrow grown in the presence of 10–6 M hydrocortisone
 

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  		Table 4. The effect of various growth factors on the blast cell-binding capacity of stromal layers from MDS bone marrow grown in the absence of 10–6 M hydrocortisone
 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
Abnormalities in the in vitro growth (number of progenitors and their cytokine sensitivity) have been described in most of the patients suffering from MDS [6-8]. Some authors observed decreased progenitor production in long-term bone marrow cultures during the progression of MDS [10, 11]. In contrast to this, normal production of progenitors was observed when normal cells were plated on stromal layers from MDS patients reflecting functional integrity of stromal microenvironment in MDS [11].

Our data suggest that the stromal layers established from MDS bone marrows are capable of supporting hemopoiesis; on the other hand, they show some specific features.

In previous experiments, irrespective of the FAB classification (RA, RARS and RAEB), MDS stromal cells grew faster and confluence was reached earlier than in the normal cultures [17]. A similar tendency for CFU-F (the only stromal cell precursor for which a clonal assay is available and can also be studied in myeloproliferative disorders [19]), was observed (our unpublished data is in good agreement with the results published by Kaneko et al. [20] and Zipori et al. [21]).

The most distinct difference between normal and MDS marrow-derived stroma was that MDS stroma could also bind primitive myeloid progenitor cells without HC, while normal stroma bound CFU-BL only if cultured in the presence of HC [22].

Although we do not have a definite explanation for this finding, some assumptions can be offered. Proteoglycans are known to play an important role in the presentation of cytokines for hemopoietic progenitor cells. Proteoglycans (chondroitin sulfate, heparan-sulfate, hyaluronic acid) belong to the major constituents of extracellular matrix in hemopoietic stroma [23]. Methylprednisolone was proved to decrease the synthesis of hyaluronic acid and modified the characteristics of sulfated proteoglycans. Hyaluronic acid was found to inhibit CFU-BL binding to stroma, consequently steroid-treated stroma with its reduced hyaluronic acid content binds more CFU-BL [24, 25]. Our present data suggest that the increased binding of blast cells to MDS stroma grown in the absence of HC may be a part of the complex dysregulation of hemopoiesis in MDS.

In our previous experiments, the stroma of aplastic anemia patients showed similar differences to MDS stroma in both growth characteristics and blast cell-binding capacity [26]. In aplastic anemia, an increased number of cortisone receptors of bone marrow cells has been suggested [27]. An increased number of cortisone receptors in MDS stromal cells, however, has not been published yet, and in spite of the presence of steroid receptors, fetal liver-derived stroma fails to bind CFU-BL [28].

Recent observations suggested that stroma derived from MDS and AML leukemia patients retains the capacity to respond to cytokine stimulation [12]. The results presented in this paper revealed that short-term incubation of the stroma with IL-3, G-CSF and SCF (c-kit ligand) increased the colony formation of CFU-BL on MDS stroma, but has no effect on normal stroma. Some authors have reported that SCF, as a synergistic factor, improved colony formation by MDS progenitors [29, 30]. In long-term bone marrow cultures, however, SCF did not result in higher progenitor yield [31]. Our present findings suggest that binding of blast cells by normal bone marrow stroma is either not directly mediated by cytokines, or in a normal situation the equilibrium of the cytokine network cannot be easily altered by added cytokines.

Both aplastic anemia and MDS are clonal disorders often transformed into acute leukemia. The similarities in the behavior of stroma from patients with aplastic anemia and MDS may point to some role of the stroma in the development of leukemia. The fact that stroma from patients with acute leukemia does not show these features [32] does not per se contradict this assumption.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
The authors wish to express their thanks to Ms. E. Kovács, Ms. O. Ullrich and Ms. M. Renner for skillful technical assistance, and Dr. S. Fekete for providing MDS samples. This work was supported by the National Research Foundation of the Hungarian Academy of Sciences (OTKA 194, OTKA 017737), and the Scientific Research Council of the Ministry of Welfare, Hungary (07 295/93).


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

  1. Mufti GJ, Galton DAG. Myelodysplastic syndromes: natural history and features of prognostic importance. Clin Haematol 1986;15:953-971.[Medline]

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Received March 11, 1996; accepted for publication May 22, 1996.




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