In Vitro Effect of Stem Cell Factor on Colony Growth from Acquired Severe Aplastic Anemia

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1 In Vitro Effect of Stem Cell Factor on Colony Growth from Acquired Severe Aplastic Anemia A. Bacigalupo," G. Piaggio," M. Podestd," M. R. Raffo," E. Tedone," G. Sogno," E Benvenuto,". Figari," L. Grassia," G. l? Bagnara? I? Strippoli? L. Bonsi,b M. E Brizzi,d G. C. Avanzi,d E Timeus,' U. Ramenghi,' G. Paolucci,b L. Pegoraro? V Gabutti" 'Divisione di Ematologia, Ospedale San Martino, Genova, Italy; bistituto di Istologia ed Embriologia, Centro Interdipartimentale di Ricerca sul Cancro Giorgio Prodi, Universith Bologna, Bologna, 1taly;'Istituto di Clinica Pediatrica e, ddipartimento di Scienze Biomediche e Oncologia Umana, Universith Torino, Torino, Italy Key Words. Stem cell factor Aplastic anemia Hemopoietic progenitors Abstract. In this study we review our present understanding of the effect of stem cell factor (SCF) on the in vitro growth of hemopoietic progenitors from patients with acquired severe aplastic anemia (SAA). We have run three separate sets of experiments. First, we have tested the expression of receptor mrnas for granulocyte-macrophage colony stimulating factor/interleukin 3 (GM-CSF/IL-3) and for c-kit protein on bone marrow (BM) cells from SAA patients. Molecular analysis revealed the presence of normal transcripts for alpha and beta chains of GM-CSFDL-3 receptor and for c-kit protein by Northern blot analysis. Second, we have tested the in vitro response to SCF of BM cells derived lrom 11 SAA patients: SCF induced a significant enhancement of erythroid burst forming unit (BFU-E) growth ( to 29, p =.1) and allowed the formation of granulocytelerythroidhnacrophsgelmegakaryocyte (GEMM) colonies which were not scored in baseline culture conditions ( to, p =.1). Granulocyte-macrophage colony forming unit (CFU-GM) growth was also enhanced (4 to 2, p =.3). This was true for patients both at diagnosis and after antilymphocyte globulin (ALG) treatment. We concluded that SCF can promote the in vitro growth of hemopoietic progenitors in patients with acquired SAA. Third, we have tested the response to SCF of peripheral blood (PB) hemopoietic progenitors Correspondence: Dr. A. Bacigalupo, Divisione Ematologia 2, Ospedale San Martino, Genova, Italy. Received March 9, 1993; accepted for publication March 9, QAlphaMed Press /93/s5./ collected from patients receiving in vivo long-term treatment with granulocyte CSF (G-CSF). When PB cells were plated directly in the presence of GM-CSF there was no colony formation. There was also no growth when plated directly in the presence of GM-CSF + IL-3 + SCF. However, after a 5-day incubation in liquid culture in the presence of GM-CSF + IL-3 + SCF there was a significant increase of CFU-GM growth from to 12,33, 11 colonies in 3/11 patients tested. These results could be duplicated by a 5-day culture in the presence of GM-CSF + leukemia inhibiting factor (LIF). These results suggest that in vitro colony growth of bone marrow and/or peripheral blood cells derived from SAA patients can be improved in the presence of SCF and are thus underestimated in standard culture conditions. The results also suggest that hemopoietic progenitors can be detected in significant numbers in the peripheral blood of these patients after prolonged in vivo use of G-CSF. Introduction Colony formation from marrow cells of patients with acquired severe aplastic anemia (SAA) is consistently low [l-41. In standard culture conditions, colony growth is unaffected by the addition of colony stimulating factors (CSFs) such as granulocyte-macrophage CSF (GM- CSF), granulocyte CSF (G-CSF), interleukin 3 (IL-3), and, only rarely, IL-1 [5]. However, in some cases of acquired aplasia, T lymphocyte depletion partially restores the in vitro response of progenitor cells to these cytokines [6]. This STEM CELLS 1993;11(~~ppl2):

2 176 Stem Cell Factor in Severe Aplastic Anemia may be taken as evidence of a quantitative defect of stem cells or of a qualitative defect of abnormalities within the accessory cells or of a combination of both. The fact that most patients with SAA recover after immunosuppressive therapy (IS) with antilymphocyte globulin (ALG) and/or cyclosporin A (CyA) [7] suggests that most patients do have an adequate stem cell reservoir, despite failure to form colonies in vitro; therefore, our in vitro culture assays would appear to be inaccurate for the enumeration of hemopoietic progenitors. This is probably also true for normal controls, but may become more evident when the number of progenitor cells is greatly reduced. Schrezenmeier and his group have recently shown with elegant experiments that the ratio of long-term initiating cells (LTIC) between aplastics and normal controls is approximately 1:lO (H. S., personal communication). We have also addressed this issue with experiments aimed at assessing the response of hemopoietic progenitors to very large doses of GM-CSF (.1 to 2, ng/ml) []. Rather than a fixed ratio, we found a different ratio between normals and aplastics, at different dose levels of GM-CSF: at very low dose (.1 ng/ml) we calculated a ratio of 1: (1 CFU-GM, grown from marrow cells of SAA patients to CFU-GM from normal controls), but at very high doses (2, ng/ml) the ratio dropped by two logs to 1: []. This would indicate that the functional state of hemopoietic progenitors is different in patients with SAA with respect to normal controls. In addition, this may lend support to the hypothesis that during pancytopenia hemopoietic progenitors are unresponsive to conventional levels of growth factors []. Stem cell factor (SCF), which stimulates more primitive hemopoietic progenitor cells in combination with other cytokines [9-1 I], can also be used to better assess the stem cell reservoir in SAA patients [12, 131. In the present report we would like to review three sets of experiments dealing with the effect of SCF on hemopoietic progenitors derived from the bone marrow or the peripheral blood of patients with aplastic anemia. Study I: mrna for GM-CSF, IL-3, SCF Receptors In order to fully evaluate the response of progenitor cells to growth factors, we first had to determine the expression of mrna for at least three cytokine receptors: GM-CSF, IL-3, and SCF. The methods used for molecular analysis have been described [12]. c-kit, GM-CSF and IL-3 receptor beta chain, GM-CSF receptor alpha chain and IL-3 receptor alpha chain transcripts were analyzed, by Northern blot analysis, in whole bone marrow samples from two SAA patients and four normal controls. To document the presence of myeloid cells in the samples, the expression of the myeloperoxidase gene was also studied. c-kir and myeloperoxydase transcripts were detected in normals as well as in both samples from SAA patients. A considerable degree of variability was observed not only among patients but also among normal bone marrow samples. Also the transcript for GM- CSF and IL-3 receptor beta chain was clearly expressed, though in variable amounts, in patients and controls. The transcript of GM- CSF and IL-3 receptor alpha chains appeared weakly expressed. However, we were able to detect transcripts for GM-CSF receptor alpha chain in both SAA patients. The expression of IL-3 receptor alpha chain gene was also expressed in variable amounts. The conclusion of this first study was that normal transcripts for alpha and beta chains of GM-CSF/IL-3 receptor and for c-kit protein were detected in marrow cells from SAA patients. Study II: Effect of SCF on Bone Marrow Cells Patients We studied 12 patients with acquired SAA, six at diagnosis, six after immunosuppressive therapy which consisted of ALG or CyA. We also studied nine healthy donors. Cell separation and colony growth conditions have been described [ 121. Colony Formation Marrow cells from patients with acquired SAA in standard culture conditions showed greatly reduced growth of CFU-GM and erythroid burst forming units (BFU-E) with respect to normal controls (Table I); granulocyte/erythroid/macrophage/megakaryocyte CFU (CFU- GEMM) growth was totally absent. The addition of SCF enhanced CFU-GM growth (p =.3) (Table I). This enhancing effect was more pronounced on BFU-E growth (JI =.1) (Table I). Finally, in five patients

3 ~ ~~ Bacigalupo et al. 177 Table I. Colony growth in vitro from non-t, non-b, non-nk light-density nonadherent mononuclear bone marrow cells in patients with SAA, FA, and normal controls Acquired SAA * 4 9 lo** 27 11** Normal controls, 9 cases Mean 16 +SD +24 CFU-GWlW BFU-EIIW CFU-GEMM/15 -SCF +SCF -SCF +SCF -SCF +SCF Abbreviation:, not done *Clonal cytogenetic abnormality 5q **MNAC, further purification was not possible I SCF allowed the formation of GEMM colonies, which were totally absent in all baseline cultures. This was seen in patients at diagnosis and after immunosuppressive treatment. Morphological examination of colonies revealed normal terminal cell differentiation. Clinical Response to Immunosuppression Out of six patients studied at diagnosis of the disease, four were treated with immunosuppression and are evaluable; all four showed enhanced colony growth with the addition of SCF. One died early of infection (case 6, day +2), two responded (cases 4.5) and one is four months post-treatment transfusion-dependent (case 9). The conclusion of this second study was that SCF is capable of enhancing the in vitro growth of hemopoietic progenitors from SAA patients, both at diagnosis and after ALG treatment. Study 111: Effect of SCF on Peripheral Blood (PB) Progenitors We have been running a pilot study within the Ematologia Bone Marrow Transplant (EBMT) aimed at reducing early mortality in SAA patients with neutrophil (PMN) counts below.5 x 19/l; this protocol includes ALG for five days, Cyk for six months, methylprednisolone (MPred) for 3 days and G-CSF 5 pg/kg/day i.v. for three months [14]. In some of the patients treated a large number of CFU- GM can be recovered and cryopreserved after at least one month of G-CSF therapy [14]. We then asked the question: can SCF enhance the growth of PB cells in cultures not showing colony growth? Table I1 outlines results in three separate experiments. PB cells were run on a Ficoll gradient, and then plated on day in the presence of GM-CSF with or without SCF, or kept in liquid culture either in GM-CSF + IL-3 + SCF or GM-CSF + leukemia inhibiting factor (LIF), and then plated in the presence of GM-CSF. We could observe no colony formation from PB cells plated on day irrespective of whether they were plated in the presence of SCF, nor on day 5 of liquid culture in the presence of GM-CSF. However, after five days of culture in the presence of SCF + IL-3 + GM-CSF there was a significant number of CFU- GM in all three patients studied. These results could be reproduced by using LIF in liquid culture in place of SCF and IL-3.

4 17 Stem Cell Factor in Severe Aplastic Anemia Table 11. Effect of SCF and LIF on peripheral blood (PB) cells derived from SAA patients after prolonged (>3 days) in vivo treatment with G-CSF Number of GM-colonies Patient 1 Patient 2 Patient 3 Baseline cultures (plated day ) +GM-CSF Baseline culture (plated day ) +GM-CSF + IL-3 + SCF Liquid culture (plated day 5) +GM-CSF Liquid culture (plated day 5 ) GM-CSF + IL-3 + SCF Liquid culture (plated day 5 ) GM-CSF + LIF Discussion Overall these results show that SCF significantly improves the in vitro colony growth from patients with acquired SAA over baseline colony formation with GM-CSF, IL-3 and erythropoietin (Epo). This is true especially for BFU-E and for CFU-GEMM, both for patients at diagnosis of the disease and after ALG treatment. It is of interest that SCF improved colony formation even in untreated patients with very severe aplastic anemia (neutrophil counts below.2 x 19/1 and platelet count below 5 x 19/1) suggesting that, in spite of severe pancytopenia and of an empty marrow, residual stem cells are still surviving. This is in line with the observation of autologous hemopoietic reconstitution following rejection of an allogenic bone marrow graft [15] and with the notion that 5% of patients treated with immunosuppressive therapy may recover to self-sustaining hematopoiesis [ 16, 171. In spite of these clinical findings, it has been so far extremely difficult to grow and quantify hemopoietic progenitors from these patients, not only at diagnosis of the disease, but also after hematologic recovery [I%]. The finding that SCF enhances the in vitro growth both with and without T cells (data not shown) suggests that the effects of SCF is not dependent on the presence/absence of accessory cells. This is in keeping with a direct effect of SCF on hemopoietic progenitors [ 191. We were also able to show that bone marrow cells derived from SAA patients express the transcripts for the alpha and beta subunits of GM-CSF/IL-3 receptor and for the c-kit protein. Preliminary results from our laboratory, obtained with an anti-c-kit antiserum, indicate that c-kit protein is expressed on CD34' cells from normal as well as from SAA bone marrow samples. It is premature to raise the question of the potential effect of SCF in vivo, although preclinical studies have shown promising results [2]. Growth factors are being used increasingly in patients with pancytopenia, although there is at present no convincing evidence of a long lasting effect on the marrow function of patients with aplastic anemia, and a transitory rise in peripheral blood counts has been the rule, with few exceptions [21-23 I. The demonstration that prolonged in vivo administration of G-CSF can mobilize a significant number of progenitor cells may be an alternative way of looking at the use of growth factors in patients with SAA; SCF would certainly be a good candidate for this purpose. Acknowledgrnen ts This work was supported by the Italian National Research Council (C.N.R.) Ematologia CT N , and by A.I.R.C. for Dr. A. Bacigalupo and by Italian Association for Cancer Research (AIRC) grants to Dr. G. P. Bagnara and Dr. L. Pegoraro. M. F. Brizzi is a fellow of AIRC. References 1 Barrett AJ, Faille A, Balitrand N. Bone marrow culture in aplastic anemia. J Clin Pathol 1979;32:66. 2 Hansi W, Rich I, Heimpel H. Erythroid colony forming cells in aplastic anemia. Br J Haematol 1977;37:43.

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