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Factor V (FV) and factor X (FX) activate and complex to form prothrombinase which subsequently cleaves prothrombin (PT), converting it to active thrombin. Thrombin cleaved osteopontin (tcOPN) contains a cryptic binding site for α4 β1 and α9 β1 integrins. We have previously shown that hematopoietic stem cells (HSC) bind to tcOPN via this site resulting in a decrease in their proliferation and differentiation. Therefore, tcOPN and the factors required for its generation are important components of the HSC niche. Herein we show mature megakaryocytes (MM, ≥8N) contain FV, FX, and PT mRNA and protein. Furthermore, we show 8N, 16N, 32N, and 64N MM all release the required factors to enable thrombin cleavage of OPN. Importantly, mice devoid of the myeloproliferative leukemia protein (Mpl), c-Mpl(-/-) mice, contain only approximately 10% of normal megakaryocyte numbers, showed significantly reduced FX and tcOPN protein levels in endosteal bone marrow (BM). In addition, WT hematopoietic progenitors and HSC showed reduced homing to the BM of c-Mpl(-/-) mice. This is the first report identifying MM as a key cellular component in the production of tcOPN in situ, allowing the BM microenvironment to self regulate HSC biology via tcOPN., (© 2015 AlphaMed Press.)

It is now evident that hemopoietic stem cells (HSC) are located in close proximity to bone lining cells within the endosteum. Accordingly, it is unlikely that the traditional method for harvesting bone marrow (BM) from mice by simply flushing long bones would result in optimal recovery of HSC. With this in mind, we have developed improved methodologies based on sequential grinding and enzymatic digestion of murine bone tissue to harvest higher numbers of BM cells and HSC from the endosteal and central marrow regions. This methodology resulted in up to a sixfold greater recovery of primitive hemopoietic cells (lineage(-)Sca(+)Kit(+) [LSK] cells) and HSC as shown by transplant studies. HSC from different anatomical regions of the marrow exhibited important functional differences. Compared with their central marrow counterparts, HSC isolated from the endosteal region (a) had 1.8-fold greater proliferative potential, (b) exhibited almost twofold greater ability to home to the BM following tail vein injection and to lodge in the endosteal region, and (c) demonstrated significantly greater long-term hemopoietic reconstitution potential as shown using limiting dilution competitive transplant assays.

There is considerable interest in the use of peripheral blood progenitor cells (PBPC) for hemopoietic rescue following high dose chemotherapy. Current regimens mobilize CD34+ with variable efficacy and there remains considerable empiricism in the design of these regimens. Some involve myelosuppression, some the administration of various cytokines alone or in combination, while a combination of chemotherapy and cytokines is employed in others. Certain protocols result in mobilization within one week while in others, maximal PBPC levels occur only after several weeks. Thus, procedures required for optimal mobilization of PBPC remain to be defined. An understanding of the mechanisms responsible for mobilization may lead to the development of improved mobilization strategies. Herein we review data that explore the mechanisms involved in the mobilization of PBPC in man. These data demonstrate that mobilization is associated with marked changes in the expression and function of cell adhesion molecules (CAMs) on hemopoietic progenitor cells (HPC), suggesting that the release of HPC into the blood involves a perturbation of the adhesive interactions between these cells and the marrow stroma that, in steady-state conditions, serve to restrict HPC to the bone marrow. Downregulation of c-kit is invariably associated with successful mobilization which, when combined with data from in vitro studies, implies a key role for stem cell factor (SCF) as an orchestrator of mobilization.