Homozygously mutated ES cells from transgenic mouse blastocyts:
Our earlier work pf Group Beug showed, that mass cultures of erythroid, myeloid and multipotent progenitors can be generated from embryoid bodies grown from mouse ES cells. In contrast to fetal liver- or bone marrow –derived cells, ES cell-derived hematopoietic progenitors have a very long lifespan in culture, but remain diploid and genetically stable. Therefore, ES cells from mutant mice are an ideal source of mutant hematopoietic cells from various lineages, including very immature multipotent progenitors. Thus, we will isolate ES cells from blastocysts of transgenic- or homozygous knockout mice, starting with Stat5^Null and Stat1^-/- mice. If justified by pilot experiments, the technology platform will also isolate ES cells from other Stat mutants, e.g. those lacking Jak2/Tyk2 or expressing knocked-in Jak- or Stat mutants. Activities will include the generation of erythroid progenitors from (mutant) ES-cell-derived embryoid bodies and initial characterization of their properties and stability.

Erythroid progenitors: Groups Beug and Müllner have demonstrated that normal murine erythroid progenitors from E12.5 fetal livers proliferate in serum-free media plus Epo, SCF and the GR-ligand Dex. Within the toolbox we propose to perform initial trials to expand erythroblasts from fetal livers of mice lacking Jaks/Stats or bearing Stat mutants.

Myeloid and lymphoid progenitors: In pilot trials, we already long-term expanded mast cell progenitors from ES-cell derived embryoid bodies. Macrophage/granulocyte progenitors were expanded from Stat5^DN and wild-type mouse bone marrow or fetal livers ⁸⁶ and should thus be accessible also from ES cells. Finally, published procedures exist for expansion of preB cells from ES-cells/embryoid bodies ⁸⁷. Therefore the toolbox will work out culture conditions to expand such progenitors from mutant mouse ES cells, and assist SFB partners in their trials to generate desired mutant cell populations.

Multipotent progenitors: Several projects of this SFB are likely to require cell culture models for multipotent hematopoietic progenitors or cells with properties of pluripotent stem cells. Examples are in vitro transformation with Stat5 mutants causing multilineage leukaemia (Group Müllner, Group Moriggl) or v-Abl-induced lymphoid leukaemias in Stat5^Null mice, which can be induced in vivo but not by infection of preB cell cultures (Group Sexl). Medium-term expansion of multipotent progenitors is possible from fetal livers by more complex cytokine/hormone combinations (SCM medium; Groups Beug and Müllner). Group Beug also generated multipotent progenitor clones showing an unlimited lifespan from embryoid bodies expressing exogenous HOXB4 (ES-HCs) and we will add these to the toolbox. A second, important activity will be to develop conditions allowing induction of selective commitment of multipotent progenitors into the erythroid or myeloid lineages.

Epithelial cells: Epithelial cells form organotypic structures in 3D collagen gels, allowing detailed analysis of epithelial plasticity in response to oncogenes and recombinant cytokines. These 3D structures can be analysed in situ for morphology, migration, epithelial/mesenchymal marker expression, apoptosis and proliferation, and are suitable for biochemical analyses. An obvious need exists to establish and further develop this technology within the SFB-JakStat, since 3D cultures were shown to reflect in vivo expression patterns of key regulators much better than conventional 2D cultures on plastic. In contrast to wild-type cells, both hepatocytes and stellate cells from p19ARF-/- mice proliferate and spontaneously immortalise in culture. These p19ARF-/- hepatocytes remain diploid, express all important liver markers and, most importantly, contribute to liver regeneration by forming normal, functional hepatocytes in vivo. The toolbox will pursue the following major aims (important for Groups Beug, Eferl and Moriggl). (i) To analyse the role of Stat1, Stat3 and Stat5 in EMT and epithelial plasticity, Stat5AlfpCre, Stat1-/- mice or floxed Stat3-/- mice will be bred onto a p19ARF-/- background. Following e.g. liver-specific deletion of Stat3 we will establish cell lines. (ii) We will develop 3D conditions that allow up-scaling and serial passage of 3D epithelial cell cultures. (iii) To allow analyses of the impact of inflammatory stroma on tumour development, we will try to develop heterotypic 3D cultures to reconstitute tumour tissue in vitro in which hepatic carcinoma cells are combined with normal or tumour stroma cells such as endothelial cells, hepatic stellate cells and Kupffer cells.