Ex Vivo Expansion of Human Hematopoietic Stem Cells by a Small-Molecule Agonist of c-MPL.

Abstract 500

The thrombopoietin (TPO) signal via its receptor, myeloproliferative leukemia virus protooncogene (c-MPL), not only regulates platelet production but also plays a crucial role in the maintenance of hematopoietic stem cells (HSCs). TPO has been characterized as a key factor for human HSCs and applied to ex vivo HSC expansion and gene transduction. However, to our knowledge, the effects of small molecule c-MPL agonists on ex vivo hematopoietic stem cell expansion and long-term hematopoietic reconstitution in vivo have not been explored in detail. In this study, we assumed that some small molecule c-MPL agonists may preferentially activate signals that facilitate self-renewal of HSCs. Thus, we have screened such small-molecule compounds and identified NR-101 as a novel c-MPL agonist compound that selectively stimulates c-MPL signaling. NR-101 exhibited in vitro potency to conduct megakaryocyte differentiation at a comparable level to that of recombinant human TPO (rhTPO). We also evaluated the effects of NR-101 on the ex vivo expansion of CD34⁺ hematopoietic stem and progenitor cells from human cord blood (hCB). We cultured hCB CD34⁺ cells in serum-free medium supplemented with rhTPO or NR-101 for 7 days and analyzed the cellular phenotype of the cultured cells by flow cytometry and colony assay. Although the total number of cells cultured with NR-101 was similar to that cultured with rhTPO, the cultures with NR-101 showed >2-fold increase in the number of CD34⁺CD38^- cells and contained 2-fold more high-proliferative-potential colony-forming cells (HPP-CFCs ; >1mm in diameter) compared to those with rhTPO. Correspondingly, SCID-repopulating cells (SRCs) were increased 2.9-fold during a 7-day culture with NR-101 compared to freshly isolated CD34⁺ cells, and 2.3-fold compared to that with TPO. These data suggest that NR-101 promotes the net expansion of hematopoietic stem and progenitor cells more efficiently than TPO, the natural ligand of c-MPL. To investigate the molecular mechanism of hematopoietic stem and progenitor cell expansion mediated by NR-101, we compared the signaling cascades stimulated by NR-101 with those by rhTPO in UT7/TPO cells (a human megakaryoblastic leukemia cell line that expresses c-MPL). When UT7/TPO cells were stimulated with rhTPO, c-MPL, JAK2, STAT3, STAT5, AKT and p44/p42 MAP kinase were immediately activated by 5 min after stimulation. By contrast, in cells stimulated with NR-101, these signaling molecules except for STAT3 became active at later time point (30 min) and showed sustained activation for significantly longer periods. Of interest, NR-101 scarcely induced STAT3 activation. Gene expression profiling by quantitative real-time PCR revealed that the major targets of the TPO/c-MPL signal are similarly upregulated in UT7/TPO cells stimulated with NR-101. However, HIF-1α protein was accumulated at a higher level and expression of its downstream target genes, including VEGF and Glucose transporter genes, was upregulated more in both UT-7/TPO and hCB CD34⁺ cells stimulated with NR-101 than those with TPO. Furthermore, we examine the cell cycle status of NR-101-treated cells by detecting BrdU incorporation. The population of CD34⁺CD38^- cells in the G0/G1 phases was significantly greater in cultures with NR-101 than in those with rhTPO. These results indicated that NR-101 activates c-MPL in a manner different from TPO does. In conclusion, we have identified NR-101, a novel nonpeptidyl small-molecule compound, which exhibits a selective and sustained activation of c-MPL. The results reported here demonstrate that NR-101 is sufficient to induce the expansion of human HSCs. The approach using NR-101 will provide a wider range of options and be useful for the development of novel and efficient technologies for hematopoietic stem cell and gene therapies.