Osteopetrotic mouse stroma with thrombopoietin, c-kit ligand, and flk-2 ligand supports long-term mobilized CD34+ hematopoiesis in vitro.
ABSTRACT OP-9 cells are stromal cells derived from macrophage colony-stimulating factor (M-CSF)-deficient osteopetrotic mice. To evaluate the OP-9 capability to sustain long-term hematopoiesis, we reported the expansion of granulocyte colony-stimulating factor (G-CSF)-mobilized human peripheral blood (PB) CD34(+) cells in co-culture with murine OP-9 and MS-5 stromal cells, either transfected with various combinations of adenovectors (Ad) expressing c-kit ligand (KL) (either soluble or transmembrane form), thrombopoietin (TPO), flt-3/flk2 ligand (FL), and granulocyte-macrophage (GM)-CSF or with weekly addition of these cytokines. Expression of TPO as well as association of TPO, FL, and KL increased progenitor cell and week-6 cobblestone area forming cell (CAFC) production in all stromal co-cultures. Similar progenitor expansion was obtained by weekly addition of soluble cytokine. Five weeks of co-culture with OP9 and TPO, FL + KL resulted in the greatest expansion of progenitor cells and week-6 CAFC as measured by secondary assay on MS-5. In contrast to MS-5 and TPO or TPO + FL + KL cultures where hematopoiesis declined by week 4, progenitor as well as week-6 CAFC expansion continued for over 3 months in TPO + FL + KL OP9 cocultures. This was associated with decrease of CD14(+) macrophage production. The addition of human macrophage (M)-CSF or CD14(+) cells to the co-culture decrease progenitor and stem cell expansion; however, murine M-CSF to OP-9 co-cultures did not decrease progenitor expansion. High levels of stromal-derived factor-1 (SDF-1) production by MS-5 and low or absent production by OP-9 may account for stem cell adhesion and CAFC formation in the former cultures and the predominance of stem and progenitor cells in the nonadherent fraction in the latter cultures.
SourceAvailable from: Edo Vellenga[Show abstract] [Hide abstract]
ABSTRACT: The level of transcription factor activity critically regulates cell fate decisions such as hematopoietic stem cell self-renewal and differentiation. The balance between hematopoietic stem cell self-renewal and differentiation needs to be tightly controlled, as a shift toward differentiation might exhaust the stem cell pool, while a shift toward self-renewal might mark the onset of leukemic transformation. A number of transcription factors have been proposed to be critically involved in governing stem cell fate and lineage commitment, such as Hox transcription factors, c-Myc, Notch1, β-catenin, C/ebpα, Pu.1 and STAT5. It is therefore no surprise that dysregulation of these transcription factors can also contribute to the development of leukemias. This review will discuss the role of STAT5 in both normal and leukemic hematopoietic stem cells as well as mechanisms by which STAT5 might contribute to the development of human leukemias.01/2012; 1(1):13-22. DOI:10.4161/jkst.19316
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ABSTRACT: Novel therapeutic approaches are urgently required for multiple myeloma (MM). We used a phenotypic screening approach using co-cultures of MM cells with bone marrow stromal cells to identify compounds that overcome stromal resistance. One such compound, BRD9876, displayed selectivity over normal hematopoietic progenitors and was discovered to be an unusual ATP non-competitive kinesin-5 (Eg5) inhibitor. A novel mutation caused resistance, suggesting a binding site distinct from known Eg5 inhibitors, and BRD9876 inhibited only microtubule-bound Eg5. Eg5 phosphorylation, which increases microtubule binding, uniquely enhanced BRD9876 activity. MM cells have greater phosphorylated Eg5 than hematopoietic cells, consistent with increased vulnerability specifically to BRD9876's mode of action. Thus, differences in Eg5-microtubule binding between malignant and normal blood cells may be exploited to treat multiple myeloma. Additional steps are required for further therapeutic development, but our results indicate that unbiased chemical biology approaches can identify therapeutic strategies unanticipated by prior knowledge of protein targets. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
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ABSTRACT: FGF1 and FGF2 support hematopoietic stem and progenitor cells (HSPCs) under stress conditions. In this study, we show that fibroblast growth factor (FGF7) may be a novel niche factor for HSPC support and leukemic growth. FGF7 expression was attenuated in mouse embryonic fibroblasts (MEFs) deficient for the MED1 subunit of the Mediator transcriptional coregulator complex. When normal mouse bone marrow (BM) cells were cocultured with Med1(+/+) MEFs or BM stromal cells in the presence of anti-FGF7 antibody, the growth of BM cells and the number of long-time culture-initiating cells (LTC-ICs) decreased significantly. Anti-FGF7 antibody also attenuated the proliferation and cobblestone formation of MB1 stromal cell-dependent myeloblastoma cells. The addition of recombinant FGF7 to the coculture of BM cells and Med1(-/-) MEFs increased BM cells and LTC-ICs. FGF7 and its cognate receptor, FGFR2IIIb, were undetectable in BM cells, but MEFs and BM stromal cells expressed both. FGF7 activated downstream targets of FGFR2IIIb in Med1(+/+) and Med1(-/-) MEFs and BM stromal cells. Taken together, we propose that FGF7 supports HSPCs and leukemia-initiating cells indirectly via FGFR2IIIb expressed on stromal cells.Biochemical and Biophysical Research Communications 09/2013; DOI:10.1016/j.bbrc.2013.09.044 · 2.28 Impact Factor