Experiments on long-term murine bone marrow cultures indicate that the production and maintenance of the hematopoietic stem cell is dependent on the establishment of an adherent monolayer and a secondary repopulation of the culture with fresh marrow. In contrast, we have found that bone marrow cultures derived from the Syrian hamster do not require a repopulation step and produce stem cells that proliferate and differentiate for more than 12 wk in the absence of an adherent layer. Stem cells were grown in Fisher's medium (pH 7.0–7.2) containing 20% horse serum in a fully humidified atmosphere of 5% CO2 in air at 37 degrees C. Cultures were fed twice weekly by removal of half of the medium and supernatant cells and replacement with an equal volume of fresh medium. No hormones or exogenous growth factors were required for the maintenance of myeloid cells, monocytes, and megakaryocytes in either the adherent or suspension cells cultures.

We have found that in liquid cultures of spleen cells of adult Syrian hamsters of the F1D strain, the hematopoietic microenvironment is adequate to sustain proliferation of splenic stem cells for periods of greater than 4 mo, and permits granulocytic, monocytic, and megakaryocytic differentiation without secondary repopulation or addition of exogenous growth factors to the basic medium of RPMI 1640 plus 20% horse serum. Intimate topographical relations are established between spleen stromal cells and hematopoietic cell components of the culture is adherent "cell-producing" islets. Some of these islets are associated with multiple hematopoietic cell types such as myeloid, monocytic, and megakaryocytic cells. Other islets are associated with a single cell type such as megakaryocytes, which suggests a limited potential of some adherent stromal cells to direct the differentiation of precursor cells. Cultures of this type provide a simple and convenient model for investigation of the mechanisms controlling differentiation of hematopoietic stem cells, not only for granulocytic and monocytic cells, but for megakaryocytic cells as well.

A long-term liquid culture system of hemopoietic tissue derived from adult hamster spleen has been described. These primary liquid cultures can maintain stem cell proliferation and differentiation for more than three months without secondary repopulation. A characteristic of the liquid cultures is the formation of clusters of hemopoietic cells around adherent stromal cells. Some islands were composed exclusively of megakaryocytes and adherent cells. Isolation of these clusters of differentiating megakaryocytes and their adherent cellular substrate permitted the analysis of the morphological and ultrastructural features of the interaction between cells of megakaryocytic lineage with the adherent stroma.