Materials and Methods: GAG-derivatized, chitosan membranes were prepared in 24 well culture plates by first casting chitosan membranes from acetic acid solution, and then covalently binding the GAG component using carbodiimide chemistry. The GAGs studied were heparin, hyaluronic acid, chondroitin-4-sulfate and dermatan sulfate. Saturating GAG densities were employed in all studies. Freshly isolated CD34+ cells from UCB were seeded into culture wells at 25,000 cells/well. Cultures were conducted in IMDM supplemented with serum, FL, TPO and SCF, with half medium changes every three days. Wells were demidepopulated at regular intervals and analyzed by flow cytometry for cell number and expression of CD34 and CD41 antigens.
Results and Discussion: CD34+ content decreased substantially within the second week regardless of the surface properties. However, the immobilized GAG surfaces profoundly enhanced the total cell expansion and the expansion of megakaryocyte progenitors. Specifically, immobilized heparin showed maximum total cell expansion of around 50 fold, almost twice the number of cells compared to the plastic control at day 14. The percentage of CD41+ cells on the heparin surface was 3.5%, 25% and 15% on day 0, day 7 and day 14 respectively. Although the CD41+ antigen expression decreased from day 7 to day 14, the number of CD41+ cells increased approximately 3-fold on day 14 compared to day 7. Additionally, there was a 3.5 fold higher number of CD41+ cells on the heparin surface compared to the plastic control. Dermatan sulfate also showed promising results. The cell morphology studied under a phase contrast microscope at day 14 showed large cells (~30-35µ) representing a morphology that might be similar to megakaryocytes. This will be verified by immunofluoroscence microscopy by staining the cells with the CD41 cell surface marker. These cells were higher in number on GAG surfaces compared to the plastic control surfaces. Lefebvre et al. have reported that serum free media resulted in a two-fold increase in megakaryocyte yield compared with serum supplemented medium [3]. Current cultures employ serum free media with rh-insulin and human transferrin as suitable serum substitutes. Since megakaryocytes are polyploid and the presence of TPO induces megakaryocyte apoptosis [4] , we are also conducting studies to evaluate the DNA ploidy level and apoptosis of megakaryocyte progenitors in our culture. The results of these studies will be reported.
Conclusions: Immobilized heparin and dermatan sulfate showed substantial potential for the expansion of total cells and megakaryocytes. The use of GAGs along with an optimal cytokine combination may accelerate the application of ex vivo expanded megakaryocyte transfusion, thereby shortening the time of platelet recovery in the thrombocytopenia induced by radiotherapy and chemotherapy.
References:
1. Gupta, P., et al., Structurally Specific Heparan Sulfates Support Primitive Human Hematopoiesis by Formation of a Multimolecular Stem Cell Niche. Blood, 1998. 92(12): p. 4641-4651. 2. Madihally, S.V., A.W. Flake, and H.W.T. Matthew, Maintenance of CD34 Expression During Proliferation of CD34+ Cord Blood Cells on Glycosaminoglycan Surfaces. Stem Cells, 1999. 17(5): p. 295-305. 3. Lefebvre P, W.J., Kahn LE, Giri JG, Cohen I., Megakaryocyte ex vivo expansion potential of three hematopoietic sources in serum and serum-free medium. J Hematother, 1999. 8(2): p. 199-208. 4. Ryu, K.-H., et al., Apoptosis and megakaryocytic differentiation during ex vivo expansion of human cord blood CD34+ cells using thrombopoietin. British Journal of Haematology, 2001. 113(2): p. 470-478.