In an attempt to analyze the cellular and molecular basis of the capacity of bone marrow stromal cells to support hematopoiesis in culture, we developed a series of murine stromal cell lines from a single long-term bone marrow culture (BMC). The cytokines produced by these cells were analyzed using immunohistochemical techniques, ribonuclease protection assays (RPA) and RT-PCR. We examined the capacity of these cloned cell lines to replace primary bone marrow-derived stromal cells in long-term bone marrow cultures (LT-BMC) and sought correlations between the capacity to support hematopoiesis in culture with the production of known cytokines. These immortalized lines replicate many of the functions of the hematopoietic microenvironment. They express cytokines known to play a role in hematopoiesis. All of the lines constitutively express mRNA for PBSF (SDF-1), macrophage colony-stimulating factor (M-CSF), stem cell factor (SCF), FLT-3, thrombopoietin (TPO), interleukin 7 (IL-7), leukemia inhibitory factor (LIF), tumor necrosis factor-beta (TNF-beta), and interferon-gamma (IFN-gamma). Most lines also express granulocyte-macrophage colony-stimulating factor (GM-CSF) and G-CSF. They vary in their expression of IL-6, tumor growth factor-beta1 (TGF-beta1), TGF-beta2, and TNF-alpha. Growing these lines in the presence of cytokines that influence hematopoiesis alters the levels of cytokine message. The most striking effects were produced by TNF-alpha. In addition to the cytokine mRNAs, the cell lines express factors associated with bone formation such as osteoblast-specific factor-2 (OSF-2) and bone morphogenetic protein-1 (BMP-1). They also express the neural cell-adhesion molecule neuropilin and neurotrophic factors including nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Several of the lines can maintain hematopoiesis in culture, as measured by the continuous production of myeloid colony-forming cells (CFU-c), for months. This capacity to support hematopoiesis does not correlate with any pattern of cytokine expression. Several of these lines also support the growth of human hematopoietic cells, and human CFU-c can be detected in the cultures in which CD34(+) bone marrow cells (BMC) are cultured on murine stromal cells. No correlation between the production of any of the known cytokines and the ability to support murine hematopoiesis was detected. In addition, there was no correlation between the capacity to support murine hematopoiesis and the capacity to maintain human HSC. Despite repeated cloning, the lines remain heterogeneous and are capable of producing cells with the properties of fibroblasts, osteoblasts, adipocytes, and myoblasts. In addition to the cytokine mRNAs, the cell lines express factors associated with bone formation such as OSF-2 and BMP-1. They also express the neural cell-adhesion molecule neuropilin and neurotrophic factors including NGF and BDNF.

Pluripotential hematopoietic stem cells grow in close association with bone marrow stromal cells, which play a critical role in sustaining hematopoiesis in long-term bone marrow cultures. The mechanisms through which stromal cells act to support pluripotential hematopoietic stem cells are largely unknown. This study demonstrates that growth arrest-specific gene-6 (GAS6) plays an important role in this process. GAS6 is a ligand for the Axl (Ufo͞Ark), Sky (Dtk͞Tyro3͞Rse͞ Brt͞Tif), and Mer (Eyk) family of tyrosine kinase receptors and binds to these receptors via tandem G domains at its C terminus. After translation, GAS6 moves to the lumen of the endoplasmic reticulum, where it is extensively ␥-carboxylated. The carboxylation process is vitamin K dependent, and current evidence suggests that GAS6 must be ␥-carboxylated to bind and activate any of the cognate tyrosine kinase receptors. Here, we show that expression of GAS6 is highly correlated with the capacity of bone marrow stromal cells to support hematopoiesis in culture. Nonsupportive stromal cell lines express little to no GAS6, whereas supportive cell lines express high levels of GAS6. Transfection of the cDNA encoding GAS6 into 3T3 fibroblasts is sufficient to render this previously nonsupportive cell line capable of supporting long-term hematopoietic cultures. 3T3 cells, genetically engineered to stably express GAS6 (GAS6-3T3), produce a stromal layer that supports the generation of colony-forming units in culture (CFU-c) for up to 6 wk. Hematopoietic support by genetically engineered 3T3 is not vitamin K dependent, and soluble recombinant GAS6 does not substitute for coculturing the hematopoietic progenitors with genetically modified 3T3 cells.

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