Heterogeneous bone-marrow stromal progenitors drive myelofibrosis via a druggable alarmin axis.

Functional contributions of individual cellular components of the bone-marrow microenvironment to myelofibrosis (MF) in patients with myeloproliferative neoplasms (MPNs) are incompletely understood. We aimed to generate a comprehensive map of the stroma in MPNs/MFs on a single-cell level in murine models and patient samples. Our analysis revealed two distinct mesenchymal stromal cell (MSC) subsets as pro-fibrotic cells. MSCs were functionally reprogrammed in a stage-dependent manner with loss of their progenitor status and initiation of differentiation in the pre-fibrotic and acquisition of a pro-fibrotic and inflammatory phenotype in the fibrotic stage. The expression of the alarmin complex S100A8/S100A9 in MSC marked disease progression toward the fibrotic phase in murine models and in patient stroma and plasma. Tasquinimod, a small-molecule inhibiting S100A8/S100A9 signaling, significantly ameliorated the MPN phenotype and fibrosis in JAK2V617F-mutated murine models, highlighting that S100A8/S100A9 is an attractive therapeutic target in MPNs.

Juxtacrine signalling via Notch and ErbB receptors in the switch to fate commitment of bone marrow-derived Schwann cells.

The phenotypic instability of adult tissue-derived Schwann cell-like cells (SCLCs) as revealed upon withdrawal of glia-inducing culture supplements limits their clinical utility for cell therapy and disease modelling. We previously overcame this limitation by co-culturing bone marrow-derived SCLCs with neurons purified from developing rat and subsequently human sensory neurons such that direct contact between cell types accomplished the cell-intrinsic switch to the Schwann cell fate. Here, our search for juxtacrine instructive signals found both Notch ligands and neuregulin-1 type III localized on the surface of DRG neurons via live cell immunocytochemistry. Bypassing ligand-induced release of the Notch intracellular domain (NICD) by transient transfection of SCLCs with the pAdlox/V5-His-NICD construct was shown to upregulate ErbB2/3. Interaction of ErbB2/3 with neuregulin-1 type III (NRG1 type III) as presented on neurons then mediated the switch to the Schwann cell fate as demonstrated by expression of S100ß/p75/ Sox10/Krox20. In contrast, treatment of cocultures with γ-secretase inhibitor perturbed Notch signalling in SCLCs and consequently deterred both upregulation of ErbB2/3 and the transition to the Schwann cell fate. Taken together, juxtacrine signalling via Notch is key to the upregulation of ErbB receptors for neuregulin-driven commitment of SCLCs to the Schwann cell fate.

Rapid and efficient generation of neural progenitors from adult bone marrow stromal cells by hypoxic preconditioning.

BACKGROUND: Bone marrow stromal cells (BMSCs) are attractive as a source of neural progenitors for ex vivo generation of neurons and glia. Limited numbers of this subpopulation, however, hinder translation into autologous cell-based therapy. Here, we demonstrate rapid and efficient conditioning with hypoxia to enrich for these neural progenitor cells prior to further expansion in neurosphere culture. METHOD: Adherent cultures of BMSCs (rat/human) were subjected to 1 % oxygen for 24 h and then subcultured as neurospheres with epidermal growth factor (EGF) and basic fibroblast growth factor supplementation. Neurospheres and cell progeny were monitored immunocytochemically for marker expression. To generate Schwann cell-like cells, neurospheres were plated out and exposed to gliogenic medium. The resulting cells were co-cultured with purified dorsal root ganglia (rat) neurons and then tested for commitment to the Schwann cell fate. Fate-committed Schwann cells were subjected to in vitro myelination assay. RESULTS: Transient hypoxic treatment increased the size and number of neurospheres generated from both rat and human BMSCs. This effect was EGF-dependent and attenuated with the EGF receptor inhibitor erlotinib. Hypoxia did not affect the capacity of neurospheres to generate neuron- or glia-like precursors. Human Schwann cell-like cells generated from hypoxia-treated BMSCs demonstrated expression of S100ß /p75 and capacity for myelination in vitro. CONCLUSION: Enhancing the yield of neural progenitor cells with hypoxic preconditioning of BMSCs in vitro but without inherent risks of genetic manipulation provides a platform for upscaling production of neural cell derivatives for clinical application in cell-based therapy.