In vitro differentiation of mouse embryonic yolk sac cells

Amiela Globerson, Vivienne Woods, Loya Abel, Lawrence Morrissey, J. Scott Cairns, Tanya Kukulansky, Louis Kubai, Robert Auerbach

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

Abstract

The embryonic yolk sac is the first site in the mammalian embryo in which cells are found that can carry out cell-mediated immune functions, yet the relation of cells of this primitive hematopoietic organ to the development of the mature immune system has not been established. We have initiated a series of experiments to determine the potential of cells of the mouse yolk sac to differentiate in vitro, in order to get an insight into the development of immunocompetence in this primary population of hematopoietic stem cells. The present paper describes the conditions promoting stem-cell differentiation and provides an initial characterization of cell surface phenotypes of the cell lineages established in vitro. Yolk sac cells obtained from 10- to 13-day mouse embryos were maintained in culture for more than 18 months, giving rise to a variety of cell types belonging to the hematopoietic lineages and culminating in the establishment of long-term cell lines. Supernatants of secondary mixed leukocyte cultures were found to be an effective source of growth factors promoting the initial differentiation as well as the maintenance of these cells. Flow-cytometric analysis showed that, in contrast to freshly obtained yolk sac cells, which had no detectable Thy 1 antigen, cells expressing significant levels of Thy 1 were obtained after 1 week or more of culture. Ly 1 and Lyt 2 antigens were detected only rarely and the L3T4 (GK 1.5) antigen was never expressed. In contrast, the Ly 5 (T200) antigen, detectable on 0%–3% of freshly isolated yolk sac cells, was expressed on the majority of cultured yolk sac cells within 1 week and was present on virtually all long-term cultured yolk sac cells. Newly isolated yolk sac cells did not express any surface-associated immuno-globulin (sIgM), but sIgM was detected on a significant number of cells in 1- to 8-week yolk sac cultures. On the other hand, no antibody binding was observed with the pre-B-cell reagents 14.8 or 6C2 (GP 220) at any time. The presence of angiotensin converting enzyme (ACE), a marker of macrophage and endothelial cells, was determined both by tripeptide cleavage tests and by immunofluorescence using a monoclonal antibody against ACE. The enzyme was limited to the adherent cell population present in yolk sac cultures. Similarly, only the adherent cell population bound acetyl low density lipoprotein (acLDL) as measured by dilacLDL fluorescence. Transformed cells appeared in at least three separate cell lines after 6–8 months in culture, and they all expressed Thy 1 and Ly 1 antigens. Both Ly 5 (T200) and Ly 9 (T100) were expressed, but neither the B220 antigen nor surface immunoglobulin could be detected. On transfer to irradiated recipients, these cells developed both as solid lymphomas at the site of injection (intramuscular or subcutaneous) and as ascites tumors following intraperitoneal injection, with maintenance of the Ly 5-possitive, Thy 1.2-positive, Ly 9-positive, Ly 1-positive pheno-type. Yolk sac cells obtained from 13-day-old mouse embryos were fused with BW 5147.3 T-lymphoma cells (Thy 1.1) to generate stable yolk sac/lymphoma hybrids. Flow-cytometric analysis demonstrated the expression of Thy 1.2, Ly 1, and Lyt 2 antigens in many of these hybrids. Following selection and cloning, various clonotypes were isolated that differed in the expression of cell surface antigens. Functional and molecular analysis of the embryonic yolk-sac cell lines, tumors, and hybrids that we have established should provide new insight into the events underlying establishment of the immune defense system in the mammalian embryo.

Original languageEnglish
Pages (from-to)185-193
Number of pages9
JournalDifferentiation
Volume36
Issue number3
DOIs
StatePublished - 1 Jan 1987
Externally publishedYes

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