Pre-clinical and clinical data on hematopoietic cell expansion have shown clear differences in primitive cell output from perfused bone marrow mononuclear cell cultures and static CD34-enriched cell cultures. A Phase I study addressed the expansion of CD34+lin- cells, with emphasis on LTC- IC output, using the perfused culture environment as a model system. Manipulation of perfusion rates and known growth factor combinations and concentrations was not effective in increasing LTC-IC output. However, contact between CD34+lin- cells and stroma induced a 50-100 kD soluble activity which increased LTC-IC output in stromal-free cultures. Phase II is proposed to identify and characterize the soluble factor(s) responsible for this activity. RT-PCR will be used to examine changes in the expression of known growth factors after direct contact of CD34+lin- cells with stroma. In addition, differential mRNA display will be utilized to identify contact-induced genes, including potentially novel stem cell factors. This approach has the added benefit of potentially identifying genes involved in stem cell homing and activation. Identification of the factor(s) with LTC-IC supportive activity has important research and clinical applications for stem cell expansion and ex vivo gene therapy.
The proposed research will address the changes in mRNA expression that are induced in stem and stromal cells upon direct contact with each other. The target of this approach is a soluble factor, known to be induced, which supports the growth of LTC-IC in purified cell cultures. Such a factor would have great commercial potential in both research and clinical markets of cell expansion and gene therapy. The approach described will also yield information on other genes induced by stem- stromal cell contact, which could potentially be useful in the commercially important areas of stem cell homing and mobilization.
