Megakaryocyte ploidy and the generation of pre/proplatelets are both increased in culture by pharmacologic inhibition of myosin-II, but nonmuscle myosin-IIA (MIIA) mutations paradoxically cause MYH9-related diseases (MYH9-RD) that adversely affect platelets. In marrow, megakaryocytes extend projections into the microcirculation, where shear facilitates fragmentation to large pre/proplatelets, suggesting that fluid stresses and myosin-II activity somehow couple in platelet biogenesis. Here, in bulk shear, plateletlike particles generated from megakaryocytes are maximized at a shear stress typical of that in the microcirculation and after treatment with a myosin-II inhibitor. MIIA activity in static cells is naturally repressed through phosphorylation at Serine-1943, but shear decreases phosphorylation, consistent with MIIA activation and localization to platelet cortex. Micropipette aspiration of cells shows myosin-II accumulates at stressed sites, but its inhibition prevents such mechanoactivation and facilitates generation of CD41(+) fragments similar in size to pre/proplatelets. MYH9-RD mutants phenocopy inhibition, revealing a dominant negative effect. MIIA is diffuse in the large platelets of a MYH9-RD patient with macrothrombocytopenia and is also diffuse in normal pre/proplatelets treated with inhibitor that blocks in vitro division to small platelets. The findings explain the large platelets in MYH9-RD and the near-normal thrombocrit of patients. Myosin-II regulation thus controls platelet size and number.

Tissue microenvironments are characterized not only in terms of chemical composition but also by collective properties such as stiffness, which influences the contractility of a cell, its adherent morphology, and even differentiation. The nucleoskeletal protein lamin-A,C increases with matrix stiffness, confers nuclear mechanical properties, and influences differentiation of mesenchymal stem cells (MSCs), whereas B-type lamins remain relatively constant. Here we show in single-cell analyses that matrix stiffness couples to myosin-II activity to promote lamin-A,C dephosphorylation at Ser22, which regulates turnover, lamina physical properties, and actomyosin expression. Lamin-A,C phosphorylation is low in interphase versus dividing cells, and its levels rise with states of nuclear rounding in which myosin-II generates little to no tension. Phosphorylated lamin-A,C localizes to nucleoplasm, and phosphorylation is enriched on lamin-A,C fragments and is suppressed by a cyclin-dependent kinase (CDK) inhibitor. Lamin-A,C knockdown in primary MSCs suppresses transcripts predominantly among actomyosin genes, especially in the serum response factor (SRF) pathway. Levels of myosin-IIA thus parallel levels of lamin-A,C, with phosphosite mutants revealing a key role for phosphoregulation. In modeling the system as a parsimonious gene circuit, we show that tension-dependent stabilization of lamin-A,C and myosin-IIA can suitably couple nuclear and cell morphology downstream of matrix mechanics.

Cell migration through solid tissue often involves large contortions of the nucleus, but biological significance is largely unclear. The nucleoskeletal protein lamin-A varies both within and between cell types and was shown here to contribute to cell sorting and survival in migration through constraining micropores. Lamin-A proved rate-limiting in 3D-migration of diverse human cells that ranged from glioma and adenocarcinoma lines to primary mesenchymal stem cells (MSCs). Stoichiometry of A- to B-type lamins established an activation barrier, with high lamin-A: B producing extruded nuclear shapes post-migration. Because the juxtaposed A, B polymer assemblies respectively conferred viscous and elastic stiffness to the nucleus, sub-populations with different A: B levels sorted in 3D-migration. However, net migration was also biphasic in lamin-A, as wildtype lamin-A levels protected against stress-induced death, whereas deep knockdown caused broad defects in stress-resistance. In vivo xenografts proved consistent with A: B-based cell sorting, and intermediate A: B enhanced tumor growth. Lamins thus impede 3D migration but also promote survival against migration-induced stresses.

Self-renewal and differentiation of stem cells depend on asymmetric division and polarized motility processes that in other cell types are modulated by nonmuscle myosin-II (MII) forces and matrix mechanics. Here, mass spectrometry-calibrated intracellular flow cytometry of human hematopoiesis reveals MIIB to be a major isoform that is strongly polarized in hematopoietic stem cells and progenitors (HSC/Ps) and thereby downregulated in differentiated cells via asymmetric division. MIIA is constitutive and activated by dephosphorylation during cytokine-triggered differentiation of cells grown on stiff, endosteum-like matrix, but not soft, marrow-like matrix. In vivo, MIIB is required for generation of blood, while MIIA is required for sustained HSC/P engraftment. Reversible inhibition of both isoforms in culture with blebbistatin enriches for long-term hematopoietic multilineage reconstituting cells by 5-fold or more as assessed in vivo. Megakaryocytes also become more polyploid, producing 4-fold more platelets. MII is thus a multifunctional node in polarized division and niche sensing.

Megakaryocytes (MKs) in the marrow extend projections into blood flow and generate platelets under shear. Understanding MK differentiation and platelet production is of broad clinical importance and extends a need to augment platelet numbers in patients. Reversible but sustained inhibition of non-muscle myosin-II (NMM-II) with the drug blebbistatin increases MK polyploidization, proplatelet formation, and membrane flexibility, thereby increasing platelet generation under shear. Using a cone and plate rheometer to apply fluid shear to drug-treated MKs in bulk, platelet-like-particles (PLPs) that are collagen-I responsive can be generated with intermediate shear. The MKs naturally down-regulate NMM-IIA activity through phosphorylation of S1943, but this site proves shear sensitive, consistent with results for human platelets. Using micropipette aspiration of MKs, inhibition of NMM-IIA is found necessary to generate CD41+ fragments that approximate the size of human platelets. Localization of NMM-IIA to the fragments is modulated by S1943 as seen by unique distribution patterns resulting from specific S1943 mutations that can be abrogated by addition of blebbistatin. The approach is extended to clinically relevant mutations associated with May-Hegglin anomaly (MHA) co-expressed with wild type protein to mimic heterozygotes. As with blebbistatin inhibition of myosin, May-Hegglin mutants result in a higher frequency of fragmentation during micropipette aspiration, indicating a dominant negative effect. Immunofluorescence documents abnormal myosin aggregation in cells transfected with May-Hegglin myosin mutations compared to wild type constructs. Finally, peripheral blood from a patient with a D1414N May-Hegglin mutation is cultured to produce megakaryocytes used to support both the micropipette and immunofluorescence results. These findings reveal a phospho-switch in NMM-II, from inactive to active in the terminal stages of platelet-poiesis, and that proper myosin activity is critical to fragment size and number. Disruption of normal activity enhances fragment generation suggesting a novel mechanism in MHA: in particular, MHA thrombocytopenia results in an increased thrombocrit due to abnormally large platelets, which overcompensates for the reduction in platelet number.

Label-free quantitation and characterization of proteins by mass spectrometry (MS) is now feasible, especially for moderately expressed structural proteins such as lamins that typically yield dozens of tryptic peptides from tissue cells. Using standard cell culture samples, we describe general algorithms for quantitative analysis of peptides identified in liquid chromatography tandem mass spectrometry (LC-MS/MS). The algorithms were foundational to the discovery that the absolute stoichiometry of A-type to B-type lamins scales with tissue stiffness (Swift et al., Science 2013). Isoform dominance helps make sense of why mutations and changes with age of mechanosensitive lamin-A,C only affect “stiff” tissues such as heart, muscle, bone, or even fat, but not brain. A Peak Ratio Fingerprinting (PRF) algorithm is elaborated here through its application to lamin-A,C knockdown. After demonstrating the large dynamic range of PRF using calibrated mixtures of human and mouse lysates, we validate measurements of partial knockdown with standard cell biology analyses using quantitative immunofluorescence and immunoblotting. Optimal sets of MS-detected peptides as determined by PRF demonstrate that the strongest peptide signals are not necessarily the most reliable for quantitation. After lamin-A,C knockdown, PRF computes an invariant set of “housekeeping” proteins as part of a broader proteomic analysis that also shows the proteome of mesenchymal stem cells (MSCs) is more broadly perturbed than that of a human epithelial cancer line (A549s), with particular variation in nuclear and cytoskeletal proteins. These methods offer exciting prospects for basic and clinical studies of lamin-A,C as well as other MS-detectable proteins.

Abstract Abstract 3456 Megakaryocytes (MKs) are rare cells that generate about 1010 platelets every day, which are necessary for vascular homeostasis via clot formation and contraction. Understanding MK differentiation and platelet production is of broad clinical importance and extends to a need to augment platelet numbers in patients. Our group has demonstrated that reversible but sustained inhibition of non-muscle myosin-II (NMM-II) increases MK polyploidization, proplatelet formation, and membrane flexibility, thereby increasing platelet generation (Shin, Spinler, et al., PNAS, 2011; 108:11458–63). The terminal steps of hematopoiesis involve the coordinated maturation and migration of multipotent hematopoietic stem cells (HSCs) from the bone marrow to the perivascular niche. Bulky, polyploid MKs do not easily transmigrate into blood, but do extend tubular membrane ‘proplatelet’ projections into the bloodstream. Visualization by others has shown that blood shear stress fragments the projections into circulating platelets, motivating a controlled study of shear effects on MKs in vitro. To recapitulate this environment in vitro, a cone and plate rheometer is used to impart physiologically relevant shear stress on MEG01s, a human MK cell line. These experiments reveal that platelet-like-particle (PLP) generation is sensitive to both biomechanical and pharmacological factors namely blebbistatin inhibition of NMM-II. We demonstrate that shear stress reduces phosphor-deactivation of NMM-II heavy chain at Ser1943 to approximately 30% of the unsheared level, which indicates a restoration of NMM-II activity necessary for proper platelet function. Stimulation of rheometer generated PLP cultures with collagen-I showed aggregation and phosphatidylserine exposure (with Annexin-V binding in the presence of Ca2+). These data demonstrates that PLPs generated in this system retain some degree of functionality such that MKs exposed to shear stress and blebbistatin result in approximately 6.5 fold more PLPs than untreated MK cultures. Other groups have shown the transport and assembly of platelet organelles to be microtubule dependent and occur de novo along proplatelets (Italiano, et al, Blood, 2005; 106:4066–75). To assess partitioning and segregation of proteins from MKs in sheared membranes, we used fluorescence-imaged micro-deformation (FIMD) to monitor CD41 and NMM-IIA during micropipette aspiration. Studies of erythrocytes had already shown a rich variety of membrane component responses to membrane distension (Discher, et al, Science, 1994; 266:1032–5), but the methods have not yet been applied to MKs. Antibody labeling of MK surface CD41 shows a homogeneous intensity along the aspirated projection of membrane, but cell body shows approximately 3 fold higher intensity, suggesting an excess of CD41 in the MK. Pre-treatment with blebbistatin increases fragmentation frequency, and these fragments show a similar trend with CD41 expression. Nucleofection was used to introduce either GFP tagged WT NMM-IIA or phosphomemetic, myosin deactivating, GFP tagged NMM-IIA S1943D to assess whether pSer impacts partitioning of this cytoskeletal protein that is abundant in platelets. Both WT and S1943D NMM-IIA are seen in the aspirated cell projection, but WT NMM-IIA clearly accumulates at the leading edge of the aspirated projection and at sites of membrane fission and fragmentation, whereas S1943D remains uniformly dispersed. These findings thus underscore the central role that NMM-II heavy chain phosphorylation, and thus activity, in proplatelet formation and platelet fragmentation. Disclosures: No relevant conflicts of interest to declare.

Scars tend to be stiffer than normal tissue, which has prompted the use of stiff matrices as models of scars, but scars are also rich in fibrillar collagen-I. Here, we introduce a soft matrix embedded with distinctly fibrillar collagen type-I, and show that this is sufficient to drive bone marrow stromal cells (MSCs) into a contractile, myofibroblastic-like phenotype – 'myo-MSCs'. These cells have been reported to minimize scarring in a unique wound healing response, exemplified by their application to myocardial infarcts. Transcriptome analysis in response to matrix rigidity points to an upregulation of genes that participate in the cellular contractile machinery, notably α-smooth muscle actin (SMA), but a decreased expression of matrix protein genes for collagen types I and VI, and tenascin-C; TGFβ1 and TGFβRII, implicated in progressive fibrosis, are also downregulated. MSCs cultured on the embedded-fiber, soft matrix exhibit many similarities to cells on rigid substrates: higher SMA but lower collagen type I protein expression. Phosphorylation at serine-1943 (S1943) of non-muscle myosin IIA, which deactivates stress fiber assembly, is decreased on both fibrosis-like and rigid substrates but almost twice higher on a soft substrate. TGFβ is found to induce S1943 phosphorylation and SMA and collagen type I production. Surprisingly, inhibition of the TGFβ pathway perturbs matrix expression but not SMA, suggesting a response that is unique from those of myofibroblasts. Furthermore, these 'myo-MSCs' hint that, unlike myofibroblasts, they do not become hyper-contractile. This supports the notion that MSC engraftment into wounded tissues suppresses fibrosis, highlighting the promise of these cells in restoring normal tissue function.

RhoA plays a multifaceted role in platelet biology. During platelet development, RhoA has been proposed to regulate endomitosis, proplatelet formation, and platelet release, in addition to having a role in platelet activation. These processes were previously studied using pharmacological inhibitors in vitro, which have potential drawbacks, such as non-specific inhibition or incomplete disruption of the intended target proteins. Therefore, we developed a conditional knockout mouse model utilizing the CRE-LOX strategy to ablate RhoA, specifically in megakaryocytes and in platelets to determine its role in platelet development. We demonstrated that deleting RhoA in megakaryocytes in vivo resulted in significant macrothrombocytopenia. RhoA-null megakaryocytes were larger, had higher mean ploidy, and exhibited stiff membranes with micropipette aspiration. However, in contrast to the results observed in experiments relying upon pharmacologic inhibitors, we did not observe any defects in proplatelet formation in megakaryocytes lacking RhoA. Infused RhoA-null megakaryocytes rapidly released platelets, but platelet levels rapidly plummeted within several hours. Our evidence supports the hypothesis that changes in membrane rheology caused infused RhoA-null megakaryocytes to prematurely release aberrant platelets that were unstable. These platelets were cleared quickly from circulation, which led to the macrothrombocytopenia. These observations demonstrate that RhoA is critical for maintaining normal megakaryocyte development and the production of normal platelets.

Hematopoietic stem and progenitor cells, as well as nucleated erythroblasts and megakaryocytes, reside preferentially in adult marrow microenvironments whereas other blood cells readily cross the endothelial barrier into the circulation. Because the nucleus is the largest organelle in blood cells, we hypothesized that (i) cell sorting across microporous barriers is regulated by nuclear deformability as controlled by lamin-A and -B, and (ii) lamin levels directly modulate hematopoietic programs. Mass spectrometry-calibrated intracellular flow cytometry indeed reveals a lamin expression map that partitions human blood lineages between marrow and circulating compartments (P = 0.00006). B-type lamins are highly variable and predominate only in CD34(+) cells, but migration through micropores and nuclear flexibility in micropipette aspiration both appear limited by lamin-A:B stoichiometry across hematopoietic lineages. Differentiation is also modulated by overexpression or knockdown of lamins as well as retinoic acid addition, which regulates lamin-A transcription. In particular, erythroid differentiation is promoted by high lamin-A and low lamin-B1 expression whereas megakaryocytes of high ploidy are inhibited by lamin suppression. Lamins thus contribute to both trafficking and differentiation.

Adult stem cells and progenitors are of great interest for their clinical application as well as their potential to reveal deep sensitivities to microenvironmental factors. The bone marrow is a niche for at least two types of stem cells, and the prototype is the hematopoietic stem cell/progenitors (HSC/Ps), which have saved many thousands of patients for several decades now. In bone marrow, HSC/Ps interact functionally with marrow stromal cells that are often referred to as mesenchymal stem cells (MSCs) or derivatives thereof. Myosin and matrix elasticity greatly affect MSC function, and these mechanobiological factors are now being explored with HSC/Ps both in vitro and in vivo. Also emerging is a role for the nucleus as a mechanically sensitive organelle that is semi-permeable to transcription factors which are modified for nuclear entry by cytoplasmic mechanobiological pathways. Since therapies envisioned with induced pluripotent stem cells and embryonic stem cells generally involve in vitro commitment to an adult stem cell or progenitor, a very deep understanding of stem cell mechanobiology is essential to progress with these multi-potent cells.

Abstract Abstract 385 The small GTPase, RhoA orchestrates actin cytoskeletal dynamics, which plays a role in platelet development and function. After platelet activation, RhoA rearranges the cytoskeleton by facilitating shape change, granule release, and clot retraction. In addition, RhoA is involved in platelet development. It does this by presumably regulating cytokinesis during megakaryocyte-erythroid progenitor cell expansion and megakaryopoiesis. RhoA also regulates thrombopoiesis by coordinating end bifurcation of the proplatelet extensions to amplify preplatelet numbers and the regulation of platelet release. Previously, most studies utilized pharmacological Rho inhibitors, such as C3 exotoxin. This could potentially obfuscate the data because of the incomplete knockdown of RhoA, the non-specific disruption of closely related Rho family members, or the long incubation times that could alter platelet biology. Therefore, we developed a transgenic mouse model that knocked out RhoA only in megakaryocytes and in platelets by using a CRE-LOX strategy to further investigate the role of RhoA in platelet biology. First, mice were generated that had loxP sites flanking the 3rd exon of RhoA (RhoAfl/fl). These mice were then crossed with mice expressing CRE recombinase driven by the platelet factor 4 promoter (PF4 CRE+), thus limiting CRE expression only in megakaryocytes and in platelets. The offspring, RhoAfl/fl PF4CRE+ mice were phenotypically normal, and had normal complete blood counts, except for macrothrombocytopenia. Their platelet counts were 25 ±3% of that observed in their littermate controls, (RhoAfl/fl PF4CRE−) and their platelet size was 130 ±10% of their littermate controls. The lack of RhoA only disrupted aggregation, granule release, and clot retraction when stimulated at the lowest dosage of agonists. To determine the causes of macrothrombocytopenia in RhoAfl/fl PF4CRE+ mice, histological examination of the spleen showed that 26.0±13.3% of the megakaryocytes had pyknotic nuclei as compared to 1.0±0.5% of the controls. In the bone marrow, apoptosis was present in 6.8±3.3% of the RhoA null megakaryocytes, but only in 1.2 ±0.3% of the control megakaryocytes. Furthermore, flow cytometry revealed that megakaryocyte counts in the bone marrow were 51.5 ±4.2% lower than in that of the controls. To determine if lacking RhoA impairs normal megakaryocyte maturation, we measured DNA ploidy using propidium iodide and flow cytometry. In megakaryocytes derived from adult bone marrow or from cultured fetal livers (E13.5, 8 days culture), the RhoAfl/fl PF4CRE+ cells had higher ploidy, a lower number of 2N cells, and an increased number of 16N cells than megakaryocytes derived from control animals. Together these data show that loss of RhoA causes deficiency of megakaryocytes probably due to increased apoptosis, and also causes aberrant maturation of the surviving megakaryocyte. To analyze whether RhoA was also required for thrombopoiesis, cultured RhoA-null megakaryocytes derived from fetal livers were infused into recipient mice. The megakaryocytes lacking RhoA more rapidly release platelets during the first 3 hours post infusion than controls. However, unlike control platelets, the Rho-null platelets were essentially gone within 24 hours. We analyzed whether the increased release of knockout platelets could be due to the up-regulation of proplatelet generation since the deletion of RhoA might impair myosin activity and impair cortical tension. However, micropipette aspiration analysis, which mimics the shear forces found in the bone marrow sinusoid capillaries, showed that the RhoA-null megakaryocytes were less compliant than the controls, primarily due to their large size. These data suggest that the higher ploidy and larger sizes of RhoA knockout megakaryocytes causes them to lodge in the pulmonary capillary bed more quickly, and to rapidly release defective macrothrombocytes. In contrast, the smaller (and thereby more compliant) wild type-cell megakaryocytes fragmented more slowly into platelets through proplatelet extension. Together, our findings demonstrate that RhoA is essential for normal megakaryocyte survival, maturation, and thrombopoiesis. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 1200 Over the last few decades, it has become clear that different blood lineages have distinct deformability, and that some blood cell types become softer in maturation perhaps to facilitate trafficking from marrow through the endothelial barrier and into the circulation (Lichtman, NEJM, 1970; 283:943–8). However, any common molecular basis of this phenomenon remains unclear. Cellular deformability is determined by elasticity of the cortex and the nucleus, and the latter is dynamically regulated by changes in expression and organization of the lamins (Pajerowski… Discher, PNAS, 2007; 104:15619–24). While gene regulation of hematopoiesis has been extensively studied, roles of lamins in blood lineages are less well understood. To address this, we developed a novel protein isoform expression analysis algorithm, “mass spectrometry calibrated intracellular flow cytometry”, to quantify lamin stoichiometries in human hematopoietic stem cells and progenitors through different mature blood lineages. This approach reveals the hematopoietic lineage map of lamins, showing that lamin A varies by 4-fold, while the normally ‘constitutive’ lamin B varies by 30-fold. During differentiation, lymphoid and myeloid lineages show decreased total lamin intensity and pliable nuclei as measured by micropipette aspiration, consistent with their ability to transmigrate into circulation. In contrast, megakaryocytes (MKs) remain in marrow because their polyploid nuclei are too large and rigid, as indicated by high lamin levels; this nuclear anchorage allows MKs to extend membrane projections into blood, where shear generates circulating platelets. Maturation of MKs is further regulated by serine phosphorylation of lamin A, since overexpression of a phospho-inactive mutant leads to increased polyploidization. Erythroid lineages share the same progenitor with MKs and migrate into blood as enucleated RBCs, because of high lamin A intensity relative to B in the progenitors and stiff chromatin. Consistent with this observation, microarray analysis of primary CD34+-derived cells indicates that some key erythroid genes are strongly correlated with lamin isoforms. Functional studies indicate that lamin A overexpression increases MK and erythroid differentiation by 2-fold, while the knockdown increases migration through pores by 2-fold. Surprisingly, increasing the lamin A to B ratio by lamin B1 knockdown decreases nuclear deformability by up to 50%, highlighting the importance of lamin isoform ratios, rather than absolute expression levels, in specifying nuclear rheology and hence traffickability of blood lineages. Finally, lamin A can be transcriptionally downregulated by retinoic acid by 2-fold, consistent with its well-established role in driving myeloid differentiation from progenitors. Together, the study suggests that nuclear deformability is hierarchically programmed by differential expression of the nucleoskeletal lamin A and B isoforms during hematopoietic differentiation, which in turn influence the ability of blood cells to migrate through marrow. Disclosures: No relevant conflicts of interest to declare.

On rigid surfaces, the cytoskeleton of migrating cells is polarized, but tissue matrix is normally soft. We show that nonmuscle MIIB (myosin-IIB) is unpolarized in cells on soft matrix in 2D and also within soft 3D collagen, with rearward polarization of MIIB emerging only as cells migrate from soft to stiff matrix. Durotaxis is the tendency of cells to crawl from soft to stiff matrix, and durotaxis of primary mesenchymal stem cells (MSCs) proved more sensitive to MIIB than to the more abundant and persistently unpolarized nonmuscle MIIA (myosin-IIA). However, MIIA has a key upstream role: in cells on soft matrix, MIIA appeared diffuse and mobile, whereas on stiff matrix, MIIA was strongly assembled in oriented stress fibers that MIIB then polarized. The difference was caused in part by elevated phospho-S1943-MIIA in MSCs on soft matrix, with site-specific mutants revealing the importance of phosphomoderated assembly of MIIA. Polarization is thus shown to be a highly regulated compass for mechanosensitive migration.

Megakaryocytes (MKs) are rare cells that every day give rise to 10 10 platelets, which are necessary for vascular homeostasis via clot formation and contraction. Understanding MK differentiation is of broad clinical importance and includes a need to augment platelet numbers in patients (e.g. post-surgery). Our group has demonstrated that reversible but sustained inhibition of NMM-II (non-muscle myosin-II) increases MK polyploidization, proplatelet formation and membrane flexibility, thereby increasing platelet generation [1]. NMM-II mutants have been implicated in platelet diseases. Furthermore, while physiological inhibition of NMM-II by phosphorylation regulates the early stage MK polyploidization, it is unknown whether phosphorylation is also important in the late stage fragmentation of platelets from MKs. We have demonstrated that applying controlled shear force in vitro increases fragmentation of platelet-like particles (PLPs) from an established human MK cell line system (MEG-01), and that pharmacological inhibition of NMM-II facilitates fragmentation even further under shear. Our current efforts our focused on introducing NMM-IIA mutants into MEG-01 cells at sites implicated in phosphorylation and May-Hegglin disorder.

Abstract Abstract 2343 Non-muscle myosin-II (NMM-II) promotes cell division, membrane rigidity and adhesion to a rigid matrix, and so NMM-II activity might be predicted to be low in dormant hematopoietic stem cells (HSCs) and to increase with differentiation. Deletion of NMM-II is known to be embryonic lethal, but its role in adult HSC differentiation is not known. Recently, we showed that sustained pharmacological inhibition of NMM-II together with soft 2D matrices like the perivascular niches in marrow, rather than rigid like bone, maximizes both MK maturation and platelet generation (Shin et al., PNAS, 2011; 108:11458-63). HSCs exhibit some similarities to mature MKs in that long-term HSCs remain undivided in vivo while various progenitors and maturing cells rapidly expand in number. Here, reversible inhibition of NMM-II sustained over several cell cycles enriches long-term HSCs up to 20 fold by selective elimination of proliferating progenitors. CFSE dilution analysis indicates that inhibition of NMM-II eliminates the accumulation phase of hematopoietic progenitors and accelerates natural cell death rate by apoptosis. Interestingly, supplementation of G-CSF significantly enhances HSC survival under NMM-II inhibition and further accelerates progenitor elimination. Molecular profiling and functional analyses indicate that NMM-II isoforms play distinct roles during HSC differentiation. NMM-IIA is a marker for differentiation with significantly lower expression in HSCs than committed progenitors, which is consistent with greater membrane flexibility of HSCs measured by micropipette aspiration. In contrast, NMM-IIB is 5 fold higher in HSCs and progenitors than differentiated CD34− cells. HSC and progenitor numbers are also sensitive to matrix elasticity in a NMM-II dependent manner, with maximal expansion on soft and high-density fibronectin matrices (not collagen). However, upon NMM-II inhibition, the extent of HSC enrichment relative to multipotent progenitors is more sensitive to matrix ligand density than matrix elasticity. To identify physiological mechanisms of regulating NMM-II activity during early HSC differentiation, we investigated post-translational modifications of NMM-IIA, specifically the de-activating and isoform-specific phosphorylation at myosin Ser1943 (pS1943) in HSC and progenitors. In a phospho-specific flow cytometry approach, pS1943 level proves highest in HSCs and decreases during differentiation with Tpo and G-CSF but not SCF alone. TGF-beta inhibits the reduction of pS1943 level, consistent with TGF-beta's known role in HSC hibernation. Therefore, pS1943 level dictates HSC enrichment and parallels the dose-response to pharmacological NMM-II inhibitors. Furthermore, phospho-mimetic mutation of NMM-IIA at Ser1943 decreases cytoskeletal integrity, increases membrane flexibility, and limits matrix elasticity sensing, indicating that biophysical properties of HSCs can also be regulated by HSC-specific signaling via NMM-IIA heavy chain phosphorylation. Myosin-inhibited CD34+-derived bone marrow cells show reduced colony-forming unit progenitors in vitro, but maintain functional long-term HSCs in vivo in the marrows of xenografted mice with an added benefit to increase platelet circulation simultaneously. Therefore, myosin-II inhibition and soft, high ligand fibronectin constitutes an important ‘microenvironment mimetic’ approach to enrichment of long-term HSCs. Myosin-II is clearly a central, matrix-regulated node for HSC proliferation and differentiation. Disclosures: No relevant conflicts of interest to declare.