Feeder-free and serum-free in vitro assay for measuring the effect of drugs on acute and chronic myeloid leukemia stem/progenitor cells.

Research on chronic and acute myeloid leukemia (CML/AML) is focused on the development of novel therapeutic strategies to eliminate leukemic stem/progenitor cells that are responsible for drug resistance and disease relapse. Methods to culture hematopoietic stem/progenitor cells (HSPCs) from blood or bone marrow samples are indispensable for investigating disease pathogenesis and delineating drug responses in individual patients. A key challenge in this area is that primary leukemic cells grow poorly in culture or rapidly differentiate and lose their hematopoietic potential. Access to patient samples can also be limiting or cell numbers too low to enable large-scale assays and/or to obtain reproducible quantitative data. Here we describe a feeder cell-free and serum-free liquid culture system for the expansion of CD34+ HSPCs from CML/AML samples and healthy control tissues. Following 7 or 14 days of culture, CD34+ cells are expanded 30- to 65-fold or 400- to 800-fold, yielding a purity of ∼80% and ∼60% CD34+ cells, respectively. This system was adapted to a 96-well format to measure the sensitivity of leukemic and normal HSPCs to cytotoxic drugs after only 7 days. The assay requires only 103 cells per well to determine drug IC50 values and can be performed with uncultured and culture-expanded cells. Importantly, resulting IC50 values strongly correlate with those obtained in the classic colony-forming unit (CFU) assay. Compared with the CFU assay, this novel 96-well liquid-based assay designed specifically for leukemic and normal HSPCs is faster and simpler, with more flexible readout methods for selecting candidates for further drug development.

Thrombopoietin is a major limiting factor for selective outgrowth of human umbilical cord blood cells in non-obese diabetic/severe combined immunodeficient recipient mice.

A single dose (0.3 microg) of recombinant human thrombopoietin (TPO) was injected into sublethal irradiated non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice immediately after transplantation of 1.5 x 10(5) purified CD34+ umbilical cord blood (UCB) cells. Bone marrow (BM) was analysed for human cells by immunophenotyping and colony culture at d 35. TPO treatment produced a two- to sixfold increase in the frequency and number of human CD45+ cells. The lineage distributions among the human cells were similar irrespective of TPO treatment; however, a prominent increase was observed in CD71+GpA- cells, reflecting the proliferative stimulus provided by TPO. The frequency of immature CD34+ cells and human granulocyte-macrophage colony-forming units and erythroid burst-forming units in TPO-treated mice was similar to that of untreated mice, but their absolute numbers had increased proportionally to the increase in human cells. The results demonstrate that human TPO is a major limiting factor for multilineage outgrowth of human UCB cells in NOD/SCID mice and can be conveniently supplemented by single-dose treatment immediately after transplantation. TPO did not affect the survival of mice after transplantation and did not significantly increase the number of immature CD34+CD38- cells; secondary transplantation revealed that TPO administration also had no significant effect on long-term repopulation. The findings demonstrate that human TPO is required for proper outgrowth of human haematopoietic stem cells after transplantation. In addition, a single administration of TPO may improve the efficiency and reproducibility of the NOD/SCID mouse assay for human immature transplantable progenitor cells.

Identification and isolation of hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are defined by their ability to repopulate all of the hematopoietic lineages in vivo and sustain the production of these cells for the life span of the individual. In the absence of reliable direct markers for HSCs, their identification and enumeration depends on functional long-term, multilineage, in vivo repopulation assays. The extremely low frequency of HSCs in any tissue and the absence of a specific HSC phenotype have made their purification and characterization a highly challenging goal. HSCs and primitive hematopoietic cells can be distinguished from mature blood cells by their lack of lineage-specific markers and presence of certain other cell-surface antigens, such as CD133 (for human cells) and c-kit and Sca-1 (for murine cells). Functional analyses of purified subpopulations of primitive hematopoietic cells have led to the development of several procedures for isolating cell populations that are highly enriched in cells with in vivo stem cell activity. Simplified methods for obtaining these cells at high yield have been important to the practical exploitation of such advances. This article reviews recent progress in identifying human and mouse HSCs and current techniques for their purification.