Megakaryocyte Colony Formation According to the Origin of Hematopoietic Stem Cell

Cho, Seung Beum; Kim, Heung Sik; Kang, Chin Moo

Abstract

BACKGROUND: Development of hematopoietic growth factor made it possible to treat anemia and granulocytopenia following intensive chemotherapy and for thrombocytopenia, recently found thrombopoietin(TPO) is being applied experimentally in several countries. The megakaryocyte colony assay can assess the effect of TPO on the thrombocytopenia resulted from cancer chemotherapy or hematopoietic stem cell transplantation. In vitro colony assay procedures for detecting human erythroid and granulocyte macrophage progenitors have been in widespread use for many years. However, reproducible assay methods for human megakaryocyte progenitors have lagged considerably behind especially in Korea. Duration platelet recovery following transplantation depends on the origin of the hematopoietic cells. Usually thrombocyte recovery is delayed following cord blood stem cell transplantation because of the small amount of cells administered. This study was carried out to investigate and establish the megakaryocyte colony assay of hematopoietic stem cells obtained from the various origin of the hematopoietic stem cells with or without TPO. METHOD: Mononuclear cells of bone marrow, peripheral blood and cord blood were collected following Ficoll density gradient centrifugation and megakaryocyte colony assay was done using MegaCultTM(Stem Cell Tech. Inc., Canada). After liquifying the agarose, mononuclear cells were added and then agarose and cell mixture were dispersed into the two wells of the chamber slide. These slides were incubated for 18~21 days at 37oC, 5% CO2. The megakaryocyte colonies were detected by staining of the cells with a primary antibody to the GPIIb/IIIa antigen, secondary antibody, alkaline phosphatase and Evans Blue in order. Changes of CD34 and GPIIb/IIIa positive cells were also analysed in flask culture using flow cytometry.
RESULTS
CD34 positive cells were most abundant in the mononuclear cells of the bone marrow, meanwhile the number of CFU-GM and megakaryocyte colony were greater in the mononuclear cells of the cord blood. After administration of TPO, the cell number of megakaryocyte colony was increased dose dependently, but CFU-GM colony did not show any response to TPO. With flask culture, the cell number was decreased with or without TPO. However adding GM-CSF, IL3 and TPO to cord blood mononuclear cell, the number of the cord blood mononuclear cells was increased on the 5 th day. The amount of CD34 positive cells was increased dose dependently to TPO in one of two cord blood and one peripheral blood. The amount of GPIIb/IIIa positive cells was increased dose dependently to TPO following incubation of all the mononuclear cells.
CONCLUSION
This study revealed successful result of megakaryocyte colony assay using MegaCultTM in various kinds of mononuclear cells and suggested that TPO was useful for CFU-mega colony formation. The amount of GPIIb/IIIa positive cells was increased with TPO in the flask culture. Therefore TPO could be useful for assessment of CFU- mega, and could be applied for the in vivo and in vitro expansion of megakaryocytes and platelets.