Ex-Vivo Expansion of Megakaryocyte Progenitors.

Following Bone Marrow Transplant, thrombocytopenia and neutropenia always occur and patients require additional post transplant support in the form of platelet transfusions.

Megakaryocytes (Mk), the precursors of platelets, are contained in hematopoietic progenitor cell products but their number is variable and relatively low. The infusion of ex vivo expanded Mk precursors could be beneficial by shortening the time to platelet engraftment and therefore reducing the amount of platelet transfusion support required by bone marrow transplant patients. The objective of this project was to investigate the expansion of Mk progenitors from peripheral blood stem cell (PBSC) harvests from patients with haematological malignancies. Briefly, CD34+ cells were isolated and cultured in serum free media supplemented with thrombopoietin (TPO) and Interleukin 1 (IL-1) then incubated at 37°C /5% CO² for 8 – 12 days. Megakaryocyte progenitor analysis was accomplished using flow cytometry analysis (CD34+/41+, CD41+, CD61+) and Mk culture analysis (CFU-Mk) (Stem Cell Technologies). Mk progenitor expansion efficiency was determined as “fold expansion” of Mk progenitors produced over input levels.

After 8 days of culture, a mean expansion of 46 fold (range 1.2 – 327.0, n=10) in megakaryocytic cells (CD61+) and a 15 fold expansion (1.2 – 41.7, n=10) in megakaryocyte progenitor cells (CD34+/41+) was observed. After 12 days, a 116 fold expansion (1.5 – 286, n=7) in megakaryocytic cells (CD61+) and a 19 fold expansion (2.4 – 40, n=7) in Mk progenitors (CD34+/CD41+) was observed.

This study demonstrates that CD34+ cells can be used to effectively expand megakaryocytic cells using just two cytokines for an incubation period of 8 – 12 days. This data could be used to develop future protocols for use in clinical applications.