Immobilized Glycosaminoglycans Reduce Thrombopoietin-Induced Apoptosis during In Vitro Expansion of Megakaryocyte Precursors from Cord Blood CD34+ Cells.

Umbilical cord blood (UCB) provides a rich source of hematopoietic stem cells for transplantation after high dose chemotherapy. However, delayed platelet recovery is a serious limitation of UCB transplantation in adults. A suggested potential solution is the transfusion of expanded megakaryocyte progenitors derived from hematopoietic stem cells (HSCs). Previous work has shown that HSC proliferation and differentiation can be influenced by the use of glycosaminoglycans (GAGs). Specifically, GAGs are known to bind and modulate the activity of many cytokines and growth factors [Gupta et al. 1998, Madihally et al. 1999]. Direct GAG-receptor interactions are also believed to play a role in this modulation. Thrombopoietin (TPO), a relatively specific megakaryocyte/platelet cytokine, plays an important role in early hematopoiesis and megakaryopoiesis. However, it has been reported that TPO induces apoptosis in cells belonging to the megakaryocyte lineage [Ryu et al. 2001]. In this study, we examined the effects of various immobilized GAGs on the expansion and apoptosis of CD41+ megakaryocyte progenitors in vitro. 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. Saturating GAG surface densities were employed in all studies. The GAGs studied were heparin, hyaluronic acid, chondroitin-4-sulfate, dermatan sulfate, heparan sulfate and the GAG analogue carboxy-methyl dextran sulfate. Freshly isolated CD34+ cells from UCB were cultured in 24 well plates at a density of 25,000 cells/well using serum free media supplemented with bovine serum albumin, rh-insulin, human transferrin, FL (50 ng/ml), TPO (50 ng/ml) and SCF (10 ng/ml). Half medium changes were done twice per week. Wells were demidepopulated at days 7, 11, and 14 and simultaneous assays of viability, CD41 antigen expression, and apoptosis (via Annexin V expression) were conducted by flow cytometry. A rapid CD41+ cell expansion was observed on all GAG surfaces except chondroitin-4-sulfate from day 7 to day 11. Plastic and chondroitin-4-sulfate showed delayed CD41+ cell expansion but by day 14 the number of viable CD41+ cells on all surfaces were comparable. An initial high expansion of CD41+ cells on the carboxy-methyl dextran sulfate surface appeared to plateau between days 11 and 14. The Annexin V analysis revealed that the GAG surfaces had a substantially lower proportion of apoptotic megakaryocytes compared to the plastic and chitosan controls. In particular, heparin, chondroitin-4-sulfate and dermatan sulfate showed two fold lower levels (p<0.05) of apoptotic megakaryocytes. These results suggest that GAGs have a substantial potential to reduce the TPO induced megakaryocyte apoptosis. 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.

View largeDownload slide

Figure

View largeDownload slide

Figure

Close modal