Megakaryocyte growth and differentiation are governed by a number of growth factors, most prominently the c-mpl ligand, Thrombopoietin (TPO). Its role on hematopoietic cell expansion is not yet clearly established. In previous studies we have defined the experimental conditions for hematopoietic cell expansion in stroma-free liquid cultures in the presence of various cytokines. We have now studied the role of TPO alone or in combination with other growth factors (FLTMigand [FL], IL3, c-kit-ligand [U], and IL6) on the megakaryocyte pathway from CD34 + cord blood cells. Twenty thousand highly purified CD34 cord blood cells were cultured in 2short-term2 liquid cultures ( up to 21 days) in the presence of medium, TPO (10 U/mL -Zymogenetics-) and /or the following growth factors :Fl(50 ng/mL -Immunex-), Kl (50 ng/mL), 116(10 ng/mL), IL3(IO ng/mt), which were added alone or in various combinations at the beginning of the cultures and then replaced twice a week. At days 3-7-14 and 21 the cultures were demidepopulated by removal of one half the culture volume which was replaced by fresh medium, TPO and the other growth factors. Cells of the harvested medium were assayed for CFU- and BfU-Hkderived colony formation. TPO alone was unable to sustain CFU- and BFU-Mk for over 2 weeks. The KL or FL supplemented cultures allowed an increased output (after 21 days FL+TPO and KL+TPO output was 10 fold the input number). The IL6-supplemented cultures were less efficacious in sustaining ex vivo megakaryocyte expansion. The 113-supplemented cultures allowed a better expansion (after 21 days of cultures the 113+TPO output was 20 fold the input number). The best expansion was seen with the combination of TPO+IL3+FL (30 fold the input number after 21 days of liquid cultures). In the presence of U+TPO±FL± 113 BFU-Mk were also detectable for up to 2 weeks. These findings will help in designing an ex vivo expansion protocol for Megakaryocyte progenitors for the management of post-transplant thrombocytopenia.
|Number of pages||1|
|Publication status||Published - 1997|
ASJC Scopus subject areas
- Cancer Research
- Cell Biology