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    • br Materials and methods br Results br Discussion We

    2018-11-08


    Materials and methods
    Results
    Discussion We report that while MM are randomly distributed within BM, 15-h post-transplant, HSC (LSKSLAM cells) preferentially home to be within two RVX-208 Supplier of MM. To investigate whether these two cell types could influence the biology of the other, we refined a FACS based method for prospectively isolating CD41+ MM, yielding a significant number of pure, viable 8N, 16N, 32N and 64N cells, allowing the assessment of MM and HSC interactions in vitro. Sorted MM were intact and bright under phase contrast pre- and post-culture. Previous methods for isolating MM and their limitations are discussed in our detailed chapter on the prospective isolation of viable MM (Heazlewood et al., 2013). Our HSC/MM co-culture studies showed that prospectively isolated MM significantly increased the proliferation of hemopoietic cells, with an expansion of cells with multi-lineage reconstitution potential as well as differentiated hemopoietic cells. Our data also showed that the MM stimulatory effect of hemopoietic cell proliferation is not cell contact-dependent, but the proliferative stimulus is mediated by the additive effect of released IGF-1 and IGFBP-3, which can be inhibited by a neutralising anti-IGF-1 antibody. We also showed that HSC express IGF-1R. While a recent review highlighted a number of papers identifying IGFBP-3 as an inhibitor of proliferation (Martin & Baxter, 2011), there are numerous studies implicating IGFBP-3, both alone and in conjunction with IGF-1, in stimulating cell proliferation. IGFBP-3 can stimulate cell proliferation by activation of sphingosine kinase-1, leading to the production of sphingosine-1-phosphate and transactivation of IGF-1R (Martin et al., 2009). Furthermore, IGFBP-3 can activate IGF-1R via the phosphatidylinositol-3 kinase pathway (Conover et al., 2000). Interestingly, there is evidence supporting IGFBP-3 directly interacting with IGF-1R (Mohseni-Zadeh & Binoux, 1997). In addition, it has also been reported that IGFBP-3 independently mediates TGF-β-induced smooth muscle cell and carcinoma cell proliferation (Cohen et al., 2000; Kansra et al., 2000), with TGF-β known to be stored and released by megakaryocytes (Fava et al., 1990). Furthermore, IGFBP-3 has been shown to synergise with Jagged-1 and Delta-1 to increase HSC proliferation (Liu et al., 2003). IGFBP-3 and acid-labile subunit are able to sequester IGF-1 to increase the half-life of IGF-1 from 10min to 15h (Guler et al., 1989; Baxter, 1994). This could account for the non-significant increase in proliferation when eLSKSLAM were cultured in IGF-1 alone; without IGFBP-3 to prolong the activity of IGF-1, eLSKSLAM received no additional signal to increase their proliferation. However, in the presence of IGF-1 and IGFBP-3, both IGF-1 direct and indirect mechanisms appear to induce hemopoietic cell proliferation. Whilst we demonstrate that IGF-1 and IGFBP-3 increase hemopoietic cell proliferation, we cannot exclude the role of other IGF and IGFBP family members. For example, IGFBP-2, -4, -5 and -6 in conjunction with, or independent of IGFs may regulate hemopoietic stem and progenitor cell migration and/or proliferation (Grellier et al., 1995; Bartling et al., 2010). Furthermore, endogenous or exogenous IGFBP-2 and IGF-2 have previously been shown to stimulate HSC proliferation (Zhang & Lodish, 2004; Huynh et al., 2008, 2011). We now demonstrate that MM release IGF-2 and IGFBP-2 into the environment and therefore may potentially regulate HSC via this mechanism. While, our IGF-1 neutralising antibody ameliorated the additive IGF-1 and IGFBP-3 induced increase in hemopoietic cell proliferation in vitro, its ability to modify the actions of other IGF and IGFBP family members remains unclear. We report that LSKSLAM cells isolated from the central BM region do not show significantly increased proliferation when co-cultured with MM, despite MM being randomly distributed throughout the marrow and HSC and MM co-localizing post-transplant. Furthermore, no difference in IGF1-R expression between LSKSLAM cells isolated from the central or endosteal region was detected (data not shown). This difference in effects on HSC isolated from the endosteal versus central BM regions is consistent with our previously published work demonstrating that although phenotypically identical for lineage, Sca-1, c-Kit, CD150 and CD48 expression, stem cells isolated from different regions of the BM have different functional potential both in vitro and in vivo (Grassinger et al., 2010; Haylock et al., 2007). These functional differences are likely to be due to a combination of intrinsic and other extrinsic microenvironmental influences. Our data demonstrates that in the presence of MM, HSC isolated from the endosteal region retain their natural ability to proliferate faster than their central counterpart. However, in the presence of MM, this increased proliferation is amplified because MM release additional stimulatory signals.