Purification and characterization of the human hemopoietic stem cell
Creators: name: Sutherland, Heather Jeanine
Publisher: name: University of British Columbia
Year: 2011

description: Previous studies in mice have suggested that some if not all hemopoietic stem cells with long-term in vivo repopulating ability are biologically, physically, and pharmacologically different from cells detectable by short-term colony assays. Since human hemopoietic stem cells cannot be assessed by expression of their in vivo repopulating potential, characterization of these cells requires an alternative endpoint. This thesis explores the use of clonogenic cell production in vitro in the presence of a competent stromal cell feeder layer for this purpose, based on the observation that this can continue for many weeks when unseparated human marrow cells are cultured under conditions that allow a stromal cell layer to form. Accordingly, a population of human clonogenic cell precursors referred to as long-term culture-initiating cells (LTC-IC) were postulated to exist as a biologically distinct compartment whose members could be quantitated by measuring the number of myeloid, erythroid and multi-lineage clonogenic progenitors present after 5 weeks of their culture on stromal feeder layers. LTC-IC in normal marrow assayed in this way were found to have a significantly lower forward light scatter, lower expression of HLA-DR, lower expression of CD 71 (transferrin receptor), and a higher expression of CD 34 as compared to clonogenic cells. Separation of marrow cells on the basis of these differences allowed a cell population enriched ~800 fold in LTC-IC to be obtained. This population contained only 0.06% of the marrow cells and 2% of the total clonogenic cells, but retained 50 - 60% of the LTC-IC present in the original marrow. Absolute numbers of LTC-IC and the proliferative and differentiative capability of individual LTC-IC were then determined by limiting dilution analysis following the demonstration that clonogenic cell output (at 5 weeks) is linearly related to input cell number over a wide range of cell concentrations. The frequency of LTC-IC in normal human marrow was determined to be ~1 per 2 x 10⁴ cells. Following purification this was increased to 1-2%. The proliferative capacity exhibited by individual LTC-IC as measured by the number of clonogenic cells per LTC-IC in 5 week-old cultures was found to range from 1 to 30 (the average being ~4). These studies also showed that a least some LTC-IC are multipotent as evident by their production of both erythroid and myeloid progeny. To study the effect of specific growth factors on LTC-IC maintenance and differentiation, highly purified LTC-IC were seeded onto irradiated murine marrow-derived stromal cells (from the M2-10B4 line) previously engineered to produce one of the human hemopoietic growth factors G-CSF, GM-CSF or IL-3. In the absence of any feeders, both the LTC-IC and their progeny in these purified suspensions decreased to very low levels within 5 weeks. However, in the presence of control M2-10B4 cells, LTC-IC maintenance and differentiation was supported as effectively as when standard human marrow feeders were present. The combined presence of G-CSF and IL-3-producing M2-10B4 cells further enhanced the maintenance and early differentiation of LTC-IC above levels obtained with control feeders, but only in the absence of GM-CSF producing feeders. In contrast, in the presence of GM-CSF-producing feeders the output of mature granulocytes and macrophages was maximal, and LTC-IC were inhibited. These studies describe and validate the use of the LTC-IC assay to selectively identify and quantitate a previously inaccessible population of very primitive human hemopoietic cells. Exploration of in vitro conditions and human growth factors able to influence their developmental behaviour points to G-CSF plus II-3 as the best candidates for future studies of LTC-IC activation and/or expansion in vitro.