Assessment of stromal function, and its potential contribution to deregulation of hematopoiesis in the myelodysplastic syndromes

Background and Objectives. The regulation of hematopoiesis by marrow stroma in vitro, has been shown to be abnormal in some patients with myelodysplas tic syndromes (MDS). This study was performed to assess whether a range of mechanisms may be altered within the MDS microenvironment. Design and Methods. The effects of diffusible factors produced by normal or MDS stromal layers on hematopoietic cells were studied by comparing the ab ility of media conditioned (CM) by normal or MDS stroma to regulate migrati on of target normal marrow CD34(+) cells across 5 mum transmembranes. The a bility of CM to stimulate hematopoietic cells was also assessed: changes in membrane polarity of KG-1a cells on exposure to stroma CM were compared. S ubsequently, contact-mediated interactions between normal marrow CD34(+) ce lls and normal and MDS stroma were studied: survival of allogeneic normal m arrow CD34(+) cells on live and glutaraldehyde-fixed normal and myelodyspla stic stroma after 24h of coculture was measured using 7-aminoactinomycin D staining. To determine whether hematopoietic cell survival on normal and MD S stroma was related to oxidative stress within the stromal microenvironmen t, intracellular superoxide levels, both constitutively and induced by tumo r necrosis factor-alpha were measured within live stromal cells by FACScan analysis of ethidium bromide stained cells. Results. The ability of CM from normal and MDS stroma to regulate short-ter m migration and activation of hematopoietic cells was similar. The mean per centage of apoptotic CD34(+) cells (13 +/- 11%) adherent to glutaraldehyde- fixed myelodysplastic stroma was higher than on paired fixed normal stroma (11 +/- 10%) (n=6, p=0.056). Constitutive mean levels of superoxide in myel odysplastic cultures (9.5 +/-2.1) were greater than in normal stromal cultu res (4.9 +/-0.6; n=6). However, following treatment with tumor necrosis fac tor-(x, the mean value for superoxide in myelodysplastic stromal cultures w as unchanged (fractional change=0.99 +/-0.56), compared with an increase in normal stroma (fractional change=1.6 +/-0.1, p <0.05). No correlation was observed between superoxide levels, proportion of apoptotic CD34+ cells and percentage of CD14(+) stromal cells [mean 8%, range 0-37% (myelodysplastic ); mean 1.3%, range 0-5% (normal)]. Interpretation and Conclusions. Abnormalities of stromal function in myelod ysplastic syndromes are likely to be heterogeneous in origin: altered matri x molecules and changes in superoxide within stromal cells may contribute t o abnormal survival and development of hematopoietic cells within the myelo dysplastic marrow microenvironment. (C) 2001, Ferrata Storti Foundation.