Poster Presentation 6th Australian Health and Medical Research Congress 2012

The role of prospectively isolated megakaryocytes in murine haemopoietic stem cell regulation (#404)

Yi Shen 1 , Brenda Williams 1 , Chad K Heazlewood 1 , Andrea Reitsma 1 , Dani Cardozo 1 , Rebecca Neaves 2 , David N Haylock 1 2 , Peter McCourt 3 , Susan K Nilsson 1 2
  1. CSIRO, Melbourne, Vic, Australia
  2. Monash University, Melbourne, Australia
  3. University of Tromsø, Tromsø, Norway

The cell fate decisions of haemopoietic stem cells (HSC) are controlled intrinsically and extrinsically by the bone marrow (BM) niche. Mature megakaryocytes (MM), responsible for platelet production, account for ~0.1-0.2% of BM cells and are randomly distributed within murine long bones. When Lin-Sca-1+Kit+CD150+CD48- HSC isolated from the endosteal region of murine long bones (eLSKSLAM) were co-cultured with SSChighCD41bright MM, we found that prospectively isolated MM were able to significantly increase the proliferation of eLSKSLAM (p<0.0001). Furthermore, the progeny maintained their haemopoietic transplant potential, giving rise to long-term multi-lineage reconstitution in vivo. When eLSKSLAM were cultured in MM conditioned media, increased eLSKSLAM proliferation was retained (p<0.001); demonstrating cell contact was not necessary. In addition, when culture media was supplemented with two factors known to be released by MM, insulin-like growth factor binding protein-3 (IGFBP-3) and insulin-like growth factor-1 (IGF-1), these factors worked synergistically to increase eLSKSLAM proliferation in a dose dependent manner; mimicking that seen in the presence of MM (p<0.001).We have also demonstrated that eLSKSLAM express the IGF-1 receptor and this increased eLSKSLAM proliferation was blocked by an anti-IGF-1 neutralising antibody (p<0.001). MM contain IGFBP-3 and IGF-1 transcripts and are also known to endocytose these factors. We recently developed a method for the prospective isolation of scavenging BM endothelial cells and identified them as an additional source of IGFBP-3. During MM maturation, DNA replication occurs without cytokinesis, resulting in polyploid cells up to 65μm in size. We have optimised a strategy for isolating MM based on ploidy and found that only when 8N, 16N and 32N MM were present in combination, and not in isolation, was there a significantly increase in eLSKSLAM proliferation. Our data suggests that MM are an important component of the HSC niche and play a role in the regulation and proliferation of HSC.