Bone marrow stroma from refractory anemia of myelodysplastic syndrome is defective in its ability to support normal CD34-positive cell proliferation and differentiation in vitro

Abstract

We examined the supportive function of stromal cells from patients with refractory anemia (RA) of myelodysplastic syndrome (MDS) on CD34-positive hematopoietic cell proliferation and differentiation using a long-term bone marrow culture (LTMC) system. Primary marrow stromal cells were obtained from 11 MDS RA patients and 12 healthy volunteers, and freshly prepared CD34-positive bone marrow cells from a normal subject were inoculated onto the stroma. There seems to be three broad patterns of hematopoietic cell growth in the LTMCs. In one group, hematopoietic cells were maintained at near normal levels (type A). In the second group, the number of hematopoietic cells increased within the first 5–10 days of culture, but declined to low levels at 15–20 days of culture as compared with normal control (type B). In the third group, the incidence of hematopoietic cells steadily declined from the beginning of the culture (type C). Furthermore, apoptotic change of hematopoietic cells was very frequently observed in cultures with the type C stroma, which were especially defective for supporting CD34+cell proliferation and differentiation. The expression of CD95 on hematopoietic cells was induced by the type C stroma, however, production of fas ligand by the stromal cells was not observed. These findings suggest a lack of hematopoietic supportive function in some cases of MDS RA and also indicate that there is heterogeneity of stromal function among MDS RA patients.

Introduction

Normal hematopoiesis is characterized by the balanced interplay between hematopoietic stem cells and the cells and molecules that form part of the microenvironment in which blood cell production takes place. This suggests that alterations in some of the elements of this microenvironment may result in the abnormal production of blood cells [1].

Myelodysplastic syndrome (MDS) is a group of acquired clonal hematopoietic disorders characterized by ineffective hematopoiesis and refractory cytopenias, and its frequent development into acute leukemia [2], [3], [4]. Quantitative and qualitative defects at the hematopoietic progenitor cell level are prominent findings in MDS [5], [6], [7], [8], [9]. Recently, Raza et al. [10] demonstrated that hematopoietic cells in bone marrow of MDS patients are highly proliferative with large numbers of erythroid, myeloid and megakaryocytic cells actively engaged in DNA synthesis. This observation is consistent with the increased cellularity of bone marrow that is usually present in MDS patients, but not with the peripheral cytopenias associated with these disorders. One possible explanation for this paradox is that the hematopoietic cells are not exiting the bone marrow compartment because they are dying before they are released into the circulation [11]. In fact, nuclear fragmentation and budding cells have long been recognized as morphological features of MDS. These cell changes have recently been identified as characteristics of cells undergoing apoptotic cell death using the technique of in situ end-labeling of fragmented DNA [11], however, it remains unclear whether the induction of apoptosis is initiated by the hematopoietic cell itself, by the stromal microenvironment (with the hematopoietic cells being the victims of a primary stromal abnormality), or by both the cell and its environment.

In long-term bone marrow cultures (LTMCs), a deficiency in the maintenance and growth of hematopoietic cells has been demonstrated [12], although no significant abnormalities in the stromal compartment have been observed [13]. Recently, functional abnormalities of MDS stromal cells have been reported, for example dysregulated cytokine production [14], [15]. Coutinho et al. [16] studied the supportive function of MDS-derived stromal cells in the proliferation and differentiation of hematopoietic stem cells that originated from normal bone marrow, using LTMC, and showed that 13 of 19 MDS stroma examined had reduced supportive activity, whereas the others were able to maintain the hematopoietic cells at near normal levels for up to 7 weeks. These observations strongly suggest that in MDS the stroma has functional abnormalities, however, the complexity of MDS populations (with refractory anemia (RA), RA with ring sideroblast, RA with excess of blasts, RA with excess of blasts in transformation and chronic myelomonocytic leukemia) makes it difficult to study the nature of the action of MDS stromal cells on hematopoietic cells.

In this experiment, we examined stromal cells from MDS patients with RA, to determine whether they have an altered ability to support hematopoiesis. Hematopoietic stem cells (CD34-positive [CD34+] cells) from normal bone marrow were cocultured with MDS RA stromal cells, and fluctuations in the number of hematopoietic cells, including granulocyte-macrophage progenitor cells (CFU-GM) were studied. Furthermore, the expression of CD95 on the surface of these cells was assayed. The appearance of hematopoietic cells with fragmented DNA induced by MDS stroma in this culture was studied by the in situ end-labeling technique.