Placental hormones contribute to changes in maternal physiology, especially to changes in the blood system. Methods are described to express a placental hormone from a cloned cDNA by transfection into a mammalian cell line, to purify the hormone, and to assess the activities of the hormone in primary mouse bone marrow cell cultures. The example used in this chapter is prolactin-like protein F (PLP-F), a recently discovered mouse placental hormone that acts on the myeloid lineage. This hormone has been expressed at high levels in stably transfected Chinese hamster ovary cells. The protein is secreted from these cells after cleavage of the signal sequence and the addition of N-linked carbohydrate. A series of chromatographic steps are used to purify the protein to homogeneity, which is verified by gel electrophoresis and silver staining; the identity of the purified protein is confirmed by immunoblot analysis. Purified protein is then assayed by addition to primary bone marrow cells and scoring the growth and the differentiation of the megakaryocyte progenitor, colony forming unit-megakaryocyte.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to determine the effect of promegakaryocytopoietic murine hormone prolactin-like protein E (PLP-E) on human megakaryocytopoiesis. Human bone marrow CD34+ cells, cultured in serum-free medium with combinations of thrombopoietin (TPO), stem cell factor (SCF), Flt-3 ligand (Flt-3L), and PLP-E, were analyzed via microscopy, flow cytometry, and clonogenic assay. Unlike the situation with mouse cells, PLP-E alone did not promote human megakaryocyte (MK) differentiation, but instead synergizes with TPO to increase colony-forming unit megakaryocyte (CFU-MK), burst-forming unit erythroid (BFU-E), and and colony-forming unit granulocyte erythroid macrophage mixed (CFU-GEMM) expansion, as well as total MK production. These effects can be attributed to an increase in colony frequency, combined with a significantly greater total cell expansion induced by adding PLP-E along with TPO. The number of cells in each CFU-MK colony is an indication of the maturity of the progenitor population, with larger colonies deriving from a more immature progenitor cell. PLP-E significantly expanded immature, intermediate, and mature CFU-MK subsets at 3 days of culture, as well as the intermediate and mature subsets at day 6. PLP-E combined with TPO induced significant expansion of all CFU-MK subsets at all time points. PLP-E further increased the effect of SCF and Flt-3L on TPO-induced total cell and CFU-MK expansion.PLP-E may act as a survival factor for primitive human megakaryocytic and erythroid progenitors. It appears to preserve the highly proliferative immature fraction of the progenitor compartment but by itself does not promote total cell proliferation or human MK production. PLP-E may prove useful in combination with TPO and other cytokines for ex vivo expansion of hematopoietic progenitors to be used in a clinical setting.
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