Analyse der Differenzierung von Makrophagen aus embryonalen Stammzellen im Mausmodel

In the present work the differentiation of macrophages from murine embryonic stem cells was investigated. One of the aims was to generate genetically modified embryonic stem cell clones, which allow an in situ monitoring for maturation and activation of macrophages. The embryonic stem cells were transfected with reporter constructs controlled by promoters of macrophage specific genes such as c-fms, Macrosialin, CD14 and iNOS. The reporter gene was EGFP, encoding a green fluorescent protein, detectable by UV-light when the promoter is active. The activity of the constructs was assayed by transient transfection of the murine macrophage cell line RAW264.7. Then the constructs were stably transfected into the murine embryonic stem cell line R1. Clones were selected via Geneticin resistance mediated by the construct and verified by PCR-Analysis. About 40% of the isolated clones had inserted the particular construct. These clones were differentiated into macrophages under established conditions. One of the embryonic stem cell clones that was modified by the c-fms-promoter-reporter construct showed green fluorescence starting on day 15 of differentiation. Maturity of the differentiation process was detectable.The embryonic stem cell derived macrophages were analyzed regarding their specificity. Progenitor cells isolated from the bone marrow and differentiated in the same way were used as a reference. Similarities were found in phagocytosis, one of the main functions of macrophages, and in expression of macrophage specific markers both on protein level and at the genetic level. Significant differences were found in cell proliferation and size. A DNA-Array-Analysis showed analogy in 90% of the expression pattern but also differentially expressed genes involved in differentiation. Of these at least eight genes known to play a role in myeloid lineage differentiation processes were identified, e.g. Ctgf encoding the connective-tissue growth factor and Csf1, the macrophage-colony stimulating factor. This data demonstrates that embryonic stem cell derived macrophages show typical properties of macrophages but at the same time can be characterized as a special type of macrophages because of the differences specified above