Effects of bFGF (Basic Fibroblast growth factor) on the Haematopoietic Sequelae that follow transplantation

Master Thesis


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University of Cape Town

Recovery following bone marrow transplantation is associated with the reduction in the clonogenic potential of stroma-adherent CD34⁺ progenitors. The bone marrow stroma is also affected, resulting in poor support for the growth of stroma-adherent multipotent progenitors that form blastic colonies (CFU-bl). To determine the possible mechanisms of marrow damage in patients treated with peripheral blood stem cells (PBSCs) and bone marrow transplantation (BMT), we studied the effects of bFGF, a cytokine known to stimulate the survival and proliferation of fibroblasts, on the propagation of clonogenic progenitors and the marrow stroma. METHODS: In order to obtain the optimal bFGF concentrations to be used on patients' haematopoietic progenitors and bone marrow rnicroenvironment, haematologically normal individuals were studied in dose response studies. Control mononuclear cells from the Ficoll-Histopaque interface layer were divided into two aliquots: one to establish a stromal layer and to culture fibroblastic progenitors (CFU-Fs) in the presence of 0, 2 and 20 ng/ml bFGF with and without 20 ng/ml heparan sulphate (HS). The second aliquot was for the selection of the progenitor population. Stroma was quantitated by placing culture dishes on a grid containing 1mm squares and scoring the number of squares occupied by stromal layers as the percentage area covered. After 3 weeks of culture, a single cell suspension was prepared by incubation of stroma with 5% trypsin solution, and number of cells in the dishes enumerated with a particle counter. CFU-Fs were terminated on the 9th day of culture, stained with May-Grilnwald-Giemsa and scored using an inverted microscope. From the second aliquot, CD34⁺ cells were incubated with paramagnetic beads and target cells isolated with a magnet. Selected l x l 04/ml cells were cultured on prefom1ed controls' stroma that has been treated with 0, 2 and 20 ng/ml bFGF. Stroma-adhered cells were covered with 0.3% agar and cultured for 6 days. Aggregates of more than 20 cells were counted as CFU-bl. RESULTS: In normal individuals, the median surface area of the petri dish covered by stroma at 3 weeks of culture was 55% (range 30-65) and was significantly improved upon the addition of 2 ng/ml (median 70%; range 50-95 ; p= <0.05) and 20 ng/ml bFGF (median 80%; range 65-99; p= 0.004). Stromal cell numbers were 0.61 x 10⁶/2ml (range 0. 15-1.66), and they increased significantly with the addition of 2 and 20 ng/ml bFGF (p=0.03). The median colony forming unit-blasts (CFU-bl) scores were 121.8 x 10⁴/ml (range 43-271), and they expanded significantly with the addition of 20 ng/ml bFGF with and without heparan sulphate (p=0.03 and 0.01). It was then concluded that the 2 and 20 ng/ml bFGF with heparan sulphate be used on patients' cells in vitro. The median surface area covered by stromal layers in patients' samples at 3 weeks was 40% (range 0-55 vs. normal 50%), and it was in1proved significantly upon the addition of bFGF (median 78 vs. 50%; p= 0.001). Supplementation of stromal layers with bFGF accounted for a significant increase in patients stromal cell numbers (2.11 vs. normal 0.61 x 106/2ml; p< 0.05). Patients' CD34⁺ cells panned on normal stromal layers resulted in significantly fewer CFU-bl (median 40 vs. normal 90 x 10⁴/ml CFU-bl; p=0.009), but CFU-bl numbers were corrected following the addition of bFGF, matching the scores achieved by normal individuals (85 vs. normal 90 x l 04/ml). Normal CD34⁺ cells proliferated poorly on patients' stromal layers in the absence of bFGF (3 9 vs. normal 90 x 10⁴/ml), but colony numbers increased significantly upon addition of bFGF (91 vs. normal 90 x 10⁴/ml CFU-bl). Thirteen patients receiving peripheral blood stem cells (PBSCs) and nine patients undergoing bone marrow transplantation (BMT) were compared. These two stem cell sources were then compared to the normal population. Stromal layers in patients receiving PBSC grafts covered a greater surface area than the area covered in patients undergoing BMT (median 65 vs. 30%). They also had higher blastic colony than scores from BMT recipients (47 vs. 12 x 10⁴/ml CFU-bl; p= 0.2). Results can be summarized that bone marrow stroma from patients receiving mobilized progenitor cells proliferates better than those receiving bone marrow grafts. It can be concluded that poor stromal layer and CD34⁺ cell proliferation following peripheral blood stem cell transplantation can be corrected by addition of basic Fibroblastic Growth Factor.