Effect of Thrombopoietin on in Vitro Production of Megakaryocytes from Fetal Mouse Liver Cells 1

Abstract

Abstract

Plasma clots containing fetal mouse liver cells (FMLC) were used to study the effects of a thrombocytopoiesis-stimulating factor (TSF) from kidney cell culture medium on the proliferation and maturation of megakaryocytes. Cells in the megakaryocytic series were identified by the presence of acetylcholinesterase (AChE) and by their morphological and ultrastructural characteristics. For these experiments, 1 × 10³ to 1 × 10⁵ FMLC were cultured for 1-7 days with 0-5 μg of TSF; control cultures were treated with production medium (PMC) in which kidney cells had not been grown. The number of AChE+ cells that were observed depended upon the number of cells plated, i.e., after 6 days of culture with 5 μg of TSF, an average of 187 AChE+ cells was found after plating 1 × 10⁴ cells and 1020 AChE+ cells were observed after plating 1 × 10⁵ cells. In dose-response experiments, the number of AChE+ cells rose with increasing doses of TSF. Significantly elevated numbers of AChE+ cells were observed after the addition of 1-5 μg of TSF. The optimum time of culture, based upon the number of AChE+ cells found, was 3-5 days. Ultrastructural analysis of megakaryocytes in plasma clots showed evidence of platelet shedding on Day 5. After the culture of FMLC with TSF, a larger number of AChE+ cells was formed from a given number of cells plated than in previous studies that used adult bone marrow cells. Therefore, because of its greater sensitivity, FMLC may be useful for the assay of low levels of TSF, and may be a valuable tool for studying the effects of megakaryocytic regulatory factors on megakaryocytopoiesis.