Effects of thrombopoietin on the proliferation and differentiation of primitive and mature haemopoietic progenitor cells in cord blood

Thrombopoietin (TPO) is considered to be the primary growth factor for regulating megakaryopoiesis and thrombopoiesis. In this study we investigated the in vitro effect of TPO on relatively immature and mature CD34+ progenitor cells in cord blood. Cells were cultured in both liquid and semi-solid cultures containing 50 ng/ml TPO. The CD34+/CD45RA- and CD34+/CD38- subfractions in cord blood were both enriched for megakaryocyte progenitors as determined in a semisolid CFU-meg assay. Progenitor cells derived from the CD34+/CD45RA- and CD34+/CD38- subfractions showed high proliferative capacity in liquid cultures. We observed a mean 19-fold expansion of the total CD34+ cell fraction, whereas in the CD34+/CD45RA- and CD34+/CD38- subfractions the mean expansion was 23- and 50-fold respectively. The expansion of the immature progenitor cell subfractions resulted in a highly purified megakaryocyte suspension containing > 80% megakaryocytes after 14 d in culture. However, these expanded megakaryocytes remained in a diploid (2N) and tetraploid (4N) state. Maturation could not be further induced by low concentration of TPO (0.1 ng/ml). The majority of the cells were 2N (80%) and 4N (15%) and only 5% of the cells had a ploidy of more than 4N. These results indicate that megakaryocyte progenitor cells in cord blood residing in the immature stem cell fraction exhibit a high proliferative capacity when cultured in the presence of TPO as the single growth factor, without maturation to hyperploid megakaryocytes.