Interestingly our data showed that when eLSKSLAM were co cul

Interestingly, our data showed that when eLSKSLAM were co-cultured with individual ploidy MM, the significant increase in proliferation was no longer observed. In addition, we observed less than expected proliferation when eLSKSLAM I-BET-762 were co-cultured with MM that had been stained with Hoechst33342 (Fig. 3G compared to Fig. 3C). This could be due to Hoechst33342 interfering with MM induced hemopoietic cell proliferation as it is known that Hoechst33342 can cause cytotoxicity and affect cell cycling (Wiezorek, 1984; Erba et al., 1988). Our experiments revealed that only 32N MM, not 8N or 16N MM, express IGFBP-3 transcript; while 8N MM express more IGF-1 transcript than 16N or 32N MM. Therefore, different ploidy populations may contribute differently to affect eLSKSLAM cell proliferation. However, ELISA data showed that compared to 32N MM, and despite having no detectable IGFBP-3 transcript, 16N MM released similar quantities of IGFBP-3 into culture media. It has previously been shown that MM endocytose and store IGF-1 and IGFBP-3 in platelet α-granules (Chan & Spencer, 1998). As we very rarely observed pro-platelet formation and platelets during MM cultures, release of these cytokines from platelets would not have impacted on our results. Taken together, it is possible that 32N MM produce endogenous IGFBP-3 as well as endocytose IGFBP-3 from the environment, and this could account for the slight but non-significant increase in hemopoietic cell proliferation while in co-culture with 32N MM. Other factors may also be involved and therefore, more experimentation is required to determine any importance of ploidy in hemopoietic cell proliferation. Our hypothesis that MM regulate HSC is also supported by several other studies. Umbilical cord blood (UCB) differentiated CD41+ megakaryocytes have a stimulatory effect on UCB stem cells by promoting the proliferation of long-term culture initiating cells (Kirouac et al., 2010). In addition, MM produce the known HSC regulator IL-6 (Suzuki et al., 1989; Jiang et al., 1994). MM also influence HSC indirectly by regulating OB (via α3β1, α5β1 and CD41), which in turn, regulate HSC (Lemieux et al., 2010). This is potentially mediated through GATA-1; because GATA-1 deficient mice have increased numbers of megakaryocytes as well as increased bone mass (Ciovacco et al., 2010). MM are a major source of factors such as thrombospondin, platelet-derived growth factor, basic fibroblast growth factor-2, bone morphogenic proteins-2, -4 and -6, receptor activator for nuclear factor κ B ligand, osteocalcin, osteonectin, osteopontin, osteoprotegerin and bone sialoprotein, which are important in the regulation of bone homeostasis, HSC, as well as recovery of the vasculature and OB following BM damage (Kacena et al., 2006; Kopp et al., 2006; Dominici et al., 2009). Therefore, the fact that megakaryocytes secrete many cytokines that influence the proliferation and survival of not only HSC and other hemopoietic cell types, but also bone formation, highlights their key regulatory role and identifies them as a potential niche component.
Authorship contributions
Disclosure of conflict of interest
Acknowledgments This work was supported by grants from the Australian Stem Cell Centre to S.N. We thank Chad Heazlewood, Andrea Reitsma and Songhui Li for technical assistance, Daniela Cardozo for assistance with animal work, Robert Baxter for intellectual input, Dave Winkler for assistance with statistical analysis, Peter McCourt for critically assessing the manuscript, Patrick Tam for RFP mice and Jochen Grassinger for providing image 1C. We also thank Andrew Fryga, Michael Reitsma, Kathryn Flanagan and Karen Clarke for flow cytometric support.
Introduction Mesenchymal stromal cells were originally described as stromal cells from bone marrow in the hematopoietic microenvironment that formed adherent colonies when cultured ex vivo and demonstrate osteogenic potential (Friedenstein et al., 1968, 1970; Sensebe et al., 2010). Since their first description, cells with similar characteristics have been derived from numerous tissues including cord blood, adipose tissue, cartilage, dental pulp, and muscle (Kuhn and Tuan, 2010). The cells obtained from bone marrow were named mesenchymal stem cells in 1991 by Caplan (Caplan, 1991). In 2005, the International Society for Cellular Therapy (ISCT) recommended the term multipotent mesenchymal stromal cells to be used to refer to fibroblast-like cells with a set of properties including plastic-adherence, in vitro trilineage differentiation capacity, and expression of a defined set of cell-surface antigens (Dominici et al., 2006; Horwitz et al., 2005). The ISCT\'s definition has been widely adopted although recent evidence has shown that the characteristics of stromal cells vary depending on their tissue sources. Moreover, the true multipotency and self-renewing capacity of stromal cells from various tissues have not been confirmed with rigorous bioassays (Bianco et al., 2013). A particular challenge to the field has been the absence of the unique set of markers that can be used to enrich MSCs from other connective tissue cell populations and define them functionally. There is much discussion of the functional definition, nomenclature, and experimental handling of multipotent stem cells as can be observed in recent reviews (Bianco et al., 2010, 2013; Keating, 2012). In this paper the term bone marrow stromal cells (BMSCs) is used to refer to plastic adhering bone marrow-derived colonies of stromal progenitors that express a set of cell-surface phenotypes defined by ISCT. Such cells have been referred to by various names in the literature including mesenchymal stem/stromal cells (MSCs).