Myosin-II Plays Central Roles In Cell Life and Death Decisions During Adult Hematopoiesis.

Abstract 1595

Hematopoietic stem cells (HSCs) are maintained in a quiescent state in a bone marrow niche, and become activated to give rise to different types of blood cells. The hierarchical design of hematopoiesis suggests that cell division-cycle machinery of each subpopulation could be differentially regulated. Our laboratory and others have previously demonstrated that non-muscle myosin-II is one of the major cytoskeletal proteins essential for cell fate decisions via adhesion to matrix and cytokinesis, both of which influence hematopoiesis. While myosin-II is essential for embryonic development, very little is known about its roles in regulating different stages of adult hematopoiesis. To test which stages of human hematopoiesis require myosin-II, bone marrow-derived CD34⁺ cells were cultured in serum-free medium with different combinations of cytokines followed by pharmacological inhibition of myosin over several cell cycles with blebbistatin. Multi-color flow cytometry analysis reveals that myosin inhibition leads to selective elimination of progenitor subpopulations via apoptotic cell death, while long-term HSC (LT-HSC) subpopulations remain viable. Transplantation of CD34⁺-derived cells treated with blebbistatin into immuno-deficient xenograft mice (NOD/Shi-scid/IL-2R^null; NSG) shows normal LT-HSC engraftment comparable to untreated cells, producing both myeloid and lymphoid lineages. Colony-forming assays confirm dose-dependent reduction of multipotent, granulocyte-macrophage and erythroid progenitors by myosin inhibition. Under culture conditions that promote differentiation of megakaryocytic (MK) lineages, myosin inhibition produces a 3–10-fold increase of polyploid MKs in suspension cultures, whereas inhibition of other pathways that also affect contractility, including the Rho-kinase pathway, have lesser effects. Myosin-II inhibition also softens the cell cortex and enhances fragmentation upon aspiration into a micropipette. Such shear flow-induced fragmentation of distensible MK processes is known to be key to how MKs in the marrow shed platelets (plts) into permeating capillaries. To test whether myosin inhibition increases plt generation in vivo, human CD34⁺-derived cells treated with blebbistatin were transplanted intra-tibially in NSG mice. Transplantation of untreated cells produced detectable circulating human CD41⁺ plts as early as 8hrs with a peak at 48∼72hrs, followed by a complete decline at 2 wks. Blebbistatin-treated cells produce plts at a more sustained level with a slower rate of decline after a peak. Overall, myosin inhibition enhances the generation of circulating human plts per CD41⁺ cell transplanted: untreated cells yield 229 ± 114 plts/day whereas blebbistatin treatment yields 804 ± 256 plts/day. Plts from both treated and untreated human cells show activated adhesion on collagen assessed ex vivo. Together these data highlight a dual role for myosin-II in adult hematopoiesis in that: 1. Myosin-II is required for survival of hematopoietic myeloid progenitors 2. Myosin-II inhibition accelerates maturation of MKs and plt generation, while maintaining survival of LT-HSCs.