Preparation and analysis of fetal liver extracts

The aim of this work is to describe the techniques that have been used for preparation and analysis of whole fetal liver extracts destined for in uter o transplantation. Nine fetal livers between 12 and 17 weeks of gestation w ere prepared: cell counts and assessment of the hematopoietic cell viabilit y were performed on cell suspensions. Hepatocytes represented 40 to 80% of the whole cell population. The remaining cells were constituted by hematopo ietic cells (mainly erythroblasts), as well as by endothelial cells, The la tter expressed CD34 on their surface, interfering with the assessment of CD 34(+) hematopoietic cells by flow cytometry, Direct visual morphologic cont rol using alkaline phosphatase anti-alkaline phosphatase techniques was nee ded to differentiate hematopoietic from extra-hematopoietic CD34(+) cells. Between 3.0 and 34.6 x 10(6) CD34(+) viable hematopoietic cells were collec ted per fetal liver, Adequate differentiation of these cells into burst-for ming units erythroid (BFU-E), colony-forming units granulocyte-macrophage ( CFU-GM), and colony-forming units granulocyte erythroid macrophage megakary ocyte (CFU-GEMM) has been shown for each sample in clonogeneic cultures. In conclusion, fetal liver is a potential source of hematopoietic stem cells. Their numeration, based on the presence of CD34, is hampered by the expres sion of this antigen on other cells contained in the liver cell extract, in particular endothelial cells.