Gαs Uncouples Hematopoietic Stem Cell Homing and Mobilization

Defects of hematopoietic stem cell adhesion or migration generally lead to reduced homing to, and enhanced mobilization from, the bone marrow. In a recent publication in Nature, Adams et al. (2009) demonstrate that the guanine-nucleotide-binding stimulatory α subunit (Gαs) can, unexpectedly, promote both phenomena

Myeloid malignancies and the microenvironment

Research in the last few years has revealed a sophisticated interaction network between multiple bone marrow (BM) cells that regulate different hematopoietic stem cell (HSC) properties, such as proliferation, differentiation, localization and self-renewal during homeostasis. These mechanisms are essential to keep the physiological HSC numbers in check and interfere with malignant progression. Besides the identification of multiple mutations and chromosomal aberrations driving the progression of myeloid malignancies, alterations in the niche compartment recently gained attention in contributing to disease progression. Leukaemic cells can remodel the niche into a permissive environment favoring leukaemic stem cell (LSC) expansion over normal HSC maintenance, and evidence is accumulating that certain niche alterations can even induce leukaemic transformation. Relapse after chemotherapy is still a major challenge during treatment of myeloid malignancies and cure is only rarely achieved. Recent progress in the understanding of niche-imposed chemoresistance mechanisms will likely contribute to the improvement of current therapeutic strategies. This Perspective article discusses the role of different niche cells and their stage- and disease-specific roles during progression of myeloid malignancies and in response to chemotherapy.This work was supported by core support grants from Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, National Health Service Blood and Transplant, and Marie Curie Career Integration Grant No. H2020-MSCA-IF-2015-708411 (C.K.) and Grant No. ERC-2014-CoG-64765 (S.M.-F.) from Horizon 2020

Bone marrow CD169+ macrophages promote the retention of hematopoietic stem and progenitor cells in the mesenchymal stem cell niche

Hematopoietic stem cells (HSCs) reside in specialized bone marrow (BM) niches regulated by the sympathetic nervous system (SNS). Here, we have examined whether mononuclear phagocytes modulate the HSC niche. We defined three populations of BM mononuclear phagocytes that include Gr-1hi monocytes (MOs), Gr-1lo MOs, and macrophages (MΦ) based on differential expression of Gr-1, CD115, F4/80, and CD169. Using MO and MΦ conditional depletion models, we found that reductions in BM mononuclear phagocytes led to reduced BM CXCL12 levels, the selective down-regulation of HSC retention genes in Nestin+ niche cells, and egress of HSCs/progenitors to the bloodstream. Furthermore, specific depletion of CD169+ MΦ, which spares BM MOs, was sufficient to induce HSC/progenitor egress. MΦ depletion also enhanced mobilization induced by a CXCR4 antagonist or granulocyte colony-stimulating factor. These results highlight two antagonistic, tightly balanced pathways that regulate maintenance of HSCs/progenitors in the niche during homeostasis, in which MΦ cross talk with the Nestin+ niche cell promotes retention, and in contrast, SNS signals enhance egress. Thus, strategies that target BM MΦ hold the potential to augment stem cell yields in patients that mobilize HSCs/progenitors poorly

Updates on the hematologic tumor microenvironment and its therapeutic targeting.

In this review article, we present recent updates on the hematologic tumor microenvironment following the 3rd Scientific Workshop on the Haematological Tumour Microenvironment and its Therapeutic Targeting organized by the European School of Hematology, which took place at the Francis Crick Institute in London in February 2019. This review article is focused on recent scientific advances highlighted in the invited presentations at the meeting, which encompassed the normal and malignant niches supporting hematopoietic stem cells and their progeny. Given the precise focus, it does not discuss other relevant contributions in this field, which have been the scope of other recent reviews. The content covers basic research and possible clinical applications with the major therapeutic angle of utilizing basic knowledge to devise new strategies to target the tumor microenvironment in hematologic cancers. The review is structured in the following sections: (i) regulation of normal hematopoietic stem cell niches during development, adulthood and aging; (ii) metabolic adaptation and reprogramming in the tumor microenvironment; (iii) the key role of inflammation in reshaping the normal microenvironment and driving hematopoietic stem cell proliferation; (iv) current understanding of the tumor microenvironment in different malignancies, such as chronic lymphocytic leukemia, multiple myeloma, acute myeloid leukemia and myelodysplastic syndromes; and (v) the effects of therapies on the microenvironment and some opportunities to target the niche directly in order to improve current treatments

Poly(vinylidene) fluoride membranes coated by heparin/collagen layer-by-layer, smart biomimetic approaches for mesenchymal stem cell culture

[EN] The use of piezoelectric materials in tissue engineering has grown considerably since inherent bone piezoelectricity was discovered. Combinations of piezoelectric polymers with magnetostrictive nanoparticles (MNP) can be used to magnetoelectrically stimulate cells by applying an external magnetic field which deforms the magnetostrictive nanoparticles in the polymer matrix, deforming the polymer itself, which varies the surface charge due to the piezoelectric effect. Poly(vinylidene) fluoride (PVDF) is the piezoelectric polymer with the largest piezoelectric coefficients, being a perfect candidate for osteogenic differentiation. As a first approach, in this paper, we propose PVDF membranes containing magnetostrictive nanoparticles and a biomimetic heparin/ collagen layer-by-layer (LbL) coating for mesenchymal stem cell culture. PVDF membranes 20% (w/v) with and without cobalt ferrite oxide (PVDF-CFO) 10% (w/w) were produced by non-solvent induced phase separation (NIPS). These membranes were found to be asymmetric, with a smooth surface, crystallinity ranging from 65% to 61%, and an electroactive beta-phase content of 51.8% and 55.6% for PVDF and PVDF-CFO, respectively. Amine groups were grafted onto the membrane surface by an alkali treatment, confirmed by ninhydrin test and X-ray photoelectron spectroscopy (XPS), providing positive charges for the assembly of heparin/collagen layers by the LbL technique. Five layers of each polyelectrolyte were deposited, ending with collagen. Human mesenchymal stem cells (hMSC) were used to test cell response in a short-term culture (1, 3 and 7 days). Nucleus cell counting showed that LbL favored cell proliferation in PVDF-CFO over non-coated membranes.This work has been funded by the Spanish State Research Agency (AEI) and the European Regional Development Fund (ERFD) through the PID2019-106099RB-C41/AEI/10.13039/501100011033 and PID2019-106099RB-C43/AEI/10.13039/501100011033 projects and the Associate Laboratory for Green Chemistry-LAQV financed by national funds from FCT/MCTES (UIDB/50006/2020). Maria GuillotFerriols acknowledges the Spanish Government funding of her doctoral thesis through a BES-2017-080398 FPI Grant. The CIBER-BBN (CB06/01/1026) initiative is funded by the VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program. CIBER actions are financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. D.M.C is also grateful to the FCT-Fundacao para a Ciencia e Tecnologia for grant SFRH/BPD/121526/2016. Finally, the authors acknowledge funding from the Basque Government Industry and Education Department under the ELKARTEK, HAZITEK and PIBA (PIBA-2018-06) programs, respectively, also Dr. Carlos Sa (CEMUP) for assistance with the XPS analyses.Guillot-Ferriols, MT.; Rodriguez-Hernandez, J.; Correia, D.; Carabineiro, S.; Lanceros-Méndez, S.; Gómez Ribelles, JL.; Gallego Ferrer, G. (2020). Poly(vinylidene) fluoride membranes coated by heparin/collagen layer-by-layer, smart biomimetic approaches for mesenchymal stem cell culture. Materials Science and Engineering C: Materials for Biological Applications (Online). 117:1-12. https://doi.org/10.1016/j.msec.2020.111281112117Jacob, J., More, N., Kalia, K., & Kapusetti, G. (2018). Piezoelectric smart biomaterials for bone and cartilage tissue engineering. Inflammation and Regeneration, 38(1). doi:10.1186/s41232-018-0059-8Fukada, E., & Yasuda, I. (1957). On the Piezoelectric Effect of Bone. Journal of the Physical Society of Japan, 12(10), 1158-1162. doi:10.1143/jpsj.12.1158Martins, P., Lopes, A. C., & Lanceros-Mendez, S. (2014). Electroactive phases of poly(vinylidene fluoride): Determination, processing and applications. Progress in Polymer Science, 39(4), 683-706. doi:10.1016/j.progpolymsci.2013.07.006Gregorio, R. (2006). Determination of the α, β, and γ crystalline phases of poly(vinylidene fluoride) films prepared at different conditions. Journal of Applied Polymer Science, 100(4), 3272-3279. doi:10.1002/app.23137Sencadas, V., Gregorio, R., & Lanceros-Méndez, S. (2009). α to β Phase Transformation and Microestructural Changes of PVDF Films Induced by Uniaxial Stretch. Journal of Macromolecular Science, Part B, 48(3), 514-525. doi:10.1080/00222340902837527Gregorio, R., & Borges, D. S. (2008). Effect of crystallization rate on the formation of the polymorphs of solution cast poly(vinylidene fluoride). Polymer, 49(18), 4009-4016. doi:10.1016/j.polymer.2008.07.010Sencadas, V., Gregorio Filho, R., & Lanceros-Mendez, S. (2006). Processing and characterization of a novel nonporous poly(vinilidene fluoride) films in the β phase. Journal of Non-Crystalline Solids, 352(21-22), 2226-2229. doi:10.1016/j.jnoncrysol.2006.02.052Buonomenna, M. G., Macchi, P., Davoli, M., & Drioli, E. (2007). Poly(vinylidene fluoride) membranes by phase inversion: the role the casting and coagulation conditions play in their morphology, crystalline structure and properties. European Polymer Journal, 43(4), 1557-1572. doi:10.1016/j.eurpolymj.2006.12.033Ribeiro, C., Costa, C. M., Correia, D. M., Nunes-Pereira, J., Oliveira, J., Martins, P., … Lanceros-Méndez, S. (2018). Electroactive poly(vinylidene fluoride)-based structures for advanced applications. Nature Protocols, 13(4), 681-704. doi:10.1038/nprot.2017.157Liu, F., Hashim, N. A., Liu, Y., Abed, M. R. M., & Li, K. (2011). Progress in the production and modification of PVDF membranes. Journal of Membrane Science, 375(1-2), 1-27. doi:10.1016/j.memsci.2011.03.014Abzan, N., Kharaziha, M., & Labbaf, S. (2019). Development of three-dimensional piezoelectric polyvinylidene fluoride-graphene oxide scaffold by non-solvent induced phase separation method for nerve tissue engineering. Materials & Design, 167, 107636. doi:10.1016/j.matdes.2019.107636Young, T.-H., Chang, H.-H., Lin, D.-J., & Cheng, L.-P. (2010). Surface modification of microporous PVDF membranes for neuron culture. Journal of Membrane Science, 350(1-2), 32-41. doi:10.1016/j.memsci.2009.12.009Gonçalves, R., Martins, P., Correia, D. M., Sencadas, V., Vilas, J. L., León, L. M., … Lanceros-Méndez, S. (2015). Development of magnetoelectric CoFe2O4 /poly(vinylidene fluoride) microspheres. RSC Advances, 5(45), 35852-35857. doi:10.1039/c5ra04409jFernandes, M. M., Correia, D. M., Ribeiro, C., Castro, N., Correia, V., & Lanceros-Mendez, S. (2019). Bioinspired Three-Dimensional Magnetoactive Scaffolds for Bone Tissue Engineering. ACS Applied Materials & Interfaces, 11(48), 45265-45275. doi:10.1021/acsami.9b14001Hermenegildo, B., Ribeiro, C., Pérez-Álvarez, L., Vilas, J. L., Learmonth, D. A., Sousa, R. A., … Lanceros-Méndez, S. (2019). Hydrogel-based magnetoelectric microenvironments for tissue stimulation. Colloids and Surfaces B: Biointerfaces, 181, 1041-1047. doi:10.1016/j.colsurfb.2019.06.023Gonçalves, R., Martins, P., Moya, X., Ghidini, M., Sencadas, V., Botelho, G., … Lanceros-Mendez, S. (2015). Magnetoelectric CoFe2O4/polyvinylidene fluoride electrospun nanofibres. Nanoscale, 7(17), 8058-8061. doi:10.1039/c5nr00453eSilva, J. M., Reis, R. L., & Mano, J. F. (2016). Biomimetic Extracellular Environment Based on Natural Origin Polyelectrolyte Multilayers. Small, 12(32), 4308-4342. doi:10.1002/smll.201601355Costa, R. R., & Mano, J. F. (2014). Polyelectrolyte multilayered assemblies in biomedical technologies. Chemical Society Reviews, 43(10), 3453. doi:10.1039/c3cs60393hCastilla-Casadiego, D. A., Pinzon-Herrera, L., Perez-Perez, M., Quiñones-Colón, B. A., Suleiman, D., & Almodovar, J. (2018). Simultaneous characterization of physical, chemical, and thermal properties of polymeric multilayers using infrared spectroscopic ellipsometry. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 553, 155-168. doi:10.1016/j.colsurfa.2018.05.052Mhanna, R. F., Vörös, J., & Zenobi-Wong, M. (2011). Layer-by-Layer Films Made from Extracellular Matrix Macromolecules on Silicone Substrates. Biomacromolecules, 12(3), 609-616. doi:10.1021/bm1012772Billings, P. C., & Pacifici, M. (2015). Interactions of signaling proteins, growth factors and other proteins with heparan sulfate: mechanisms and mysteries. Connective Tissue Research, 56(4), 272-280. doi:10.3109/03008207.2015.1045066Chen, J., Huang, N., Li, Q., Chu, C. H., Li, J., & Maitz, M. F. (2016). The effect of electrostatic heparin/collagen layer-by-layer coating degradation on the biocompatibility. Applied Surface Science, 362, 281-289. doi:10.1016/j.apsusc.2015.11.227Zhang, K., Huang, D., Yan, Z., & Wang, C. (2017). Heparin/collagen encapsulating nerve growth factor multilayers coated aligned PLLA nanofibrous scaffolds for nerve tissue engineering. Journal of Biomedical Materials Research Part A, 105(7), 1900-1910. doi:10.1002/jbm.a.36053Ferreira, A. M., Gentile, P., Toumpaniari, S., Ciardelli, G., & Birch, M. A. (2016). Impact of Collagen/Heparin Multilayers for Regulating Bone Cellular Functions. ACS Applied Materials & Interfaces, 8(44), 29923-29932. doi:10.1021/acsami.6b09241Castilla-Casadiego, D. A., García, J. R., García, A. J., & Almodovar, J. (2019). Heparin/Collagen Coatings Improve Human Mesenchymal Stromal Cell Response to Interferon Gamma. ACS Biomaterials Science & Engineering, 5(6), 2793-2803. doi:10.1021/acsbiomaterials.9b00008Martins, P., Gonçalves, R., Lanceros-Mendez, S., Lasheras, A., Gutiérrez, J., & Barandiarán, J. M. (2014). Effect of filler dispersion and dispersion method on the piezoelectric and magnetoelectric response of CoFe2O4/P(VDF-TrFE) nanocomposites. Applied Surface Science, 313, 215-219. doi:10.1016/j.apsusc.2014.05.187Gamboa-Martínez, T. C., Luque-Guillén, V., González-García, C., Gómez Ribelles, J. L., & Gallego-Ferrer, G. (2014). Crosslinked fibrin gels for tissue engineering: Two approaches to improve their properties. Journal of Biomedical Materials Research Part A, 103(2), 614-621. doi:10.1002/jbm.a.35210Gregorio, Jr., R., & Cestari, M. (1994). Effect of crystallization temperature on the crystalline phase content and morphology of poly(vinylidene fluoride). Journal of Polymer Science Part B: Polymer Physics, 32(5), 859-870. doi:10.1002/polb.1994.090320509Martins, P., Costa, C. M., & Lanceros-Mendez, S. (2010). Nucleation of electroactive β-phase poly(vinilidene fluoride) with CoFe2O4 and NiFe2O4 nanofillers: a new method for the preparation of multiferroic nanocomposites. Applied Physics A, 103(1), 233-237. doi:10.1007/s00339-010-6003-7Qi, L., Knapton, E. K., Zhang, X., Zhang, T., Gu, C., & Zhao, Y. (2017). Pre-culture Sudan Black B treatment suppresses autofluorescence signals emitted from polymer tissue scaffolds. Scientific Reports, 7(1). doi:10.1038/s41598-017-08723-2Young, T.-H., Cheng, L.-P., Lin, D.-J., Fane, L., & Chuang, W.-Y. (1999). Mechanisms of PVDF membrane formation by immersion-precipitation in soft (1-octanol) and harsh (water) nonsolvents. Polymer, 40(19), 5315-5323. doi:10.1016/s0032-3861(98)00747-2Cheng, L.-P. (1999). Effect of Temperature on the Formation of Microporous PVDF Membranes by Precipitation from 1-Octanol/DMF/PVDF and Water/DMF/PVDF Systems. Macromolecules, 32(20), 6668-6674. doi:10.1021/ma990418lSupriya, S., Kumar, L., & Kar, M. (2018). Optimization of dielectric properties of PVDF-CFO nanocomposites. Polymer Composites, 40(3), 1239-1250. doi:10.1002/pc.24840Lin, D.-J., Beltsios, K., Young, T.-H., Jeng, Y.-S., & Cheng, L.-P. (2006). Strong effect of precursor preparation on the morphology of semicrystalline phase inversion poly(vinylidene fluoride) membranes. Journal of Membrane Science, 274(1-2), 64-72. doi:10.1016/j.memsci.2005.07.043Cai, X., Lei, T., Sun, D., & Lin, L. (2017). A critical analysis of the α, β and γ phases in poly(vinylidene fluoride) using FTIR. RSC Advances, 7(25), 15382-15389. doi:10.1039/c7ra01267eBoccaccio, T., Bottino, A., Capannelli, G., & Piaggio, P. (2002). Characterization of PVDF membranes by vibrational spectroscopy. Journal of Membrane Science, 210(2), 315-329. doi:10.1016/s0376-7388(02)00407-6Liu, J., Lu, X., & Wu, C. (2013). Effect of Preparation Methods on Crystallization Behavior and Tensile Strength of Poly(vinylidene fluoride) Membranes. Membranes, 3(4), 389-405. doi:10.3390/membranes3040389ZHANG, M., ZHANG, A., ZHU, B., DU, C., & XU, Y. (2008). Polymorphism in porous poly(vinylidene fluoride) membranes formed via immersion precipitation process. Journal of Membrane Science, 319(1-2), 169-175. doi:10.1016/j.memsci.2008.03.029Xiao, L., Davenport, D. M., Ormsbee, L., & Bhattacharyya, D. (2015). Polymerization and Functionalization of Membrane Pores for Water Related Applications. Industrial & Engineering Chemistry Research, 54(16), 4174-4182. doi:10.1021/ie504149tDuca, M. D., Plosceanu, C. L., & Pop, T. (1998). Effect of X-rays on poly(vinylidene fluoride) in X-ray photoelectron spectroscopy. Journal of Applied Polymer Science, 67(13), 2125-2129. doi:10.1002/(sici)1097-4628(19980328)67:133.0.co;2-gCorreia, D. M., Ribeiro, C., Sencadas, V., Botelho, G., Carabineiro, S. A. C., Ribelles, J. L. G., & Lanceros-Méndez, S. (2015). Influence of oxygen plasma treatment parameters on poly(vinylidene fluoride) electrospun fiber mats wettability. Progress in Organic Coatings, 85, 151-158. doi:10.1016/j.porgcoat.2015.03.019Kehrer, M., Duchoslav, J., Hinterreiter, A., Cobet, M., Mehic, A., Stehrer, T., & Stifter, D. (2019). XPS investigation on the reactivity of surface imine groups with TFAA. Plasma Processes and Polymers, 16(4), 1800160. doi:10.1002/ppap.201800160Morales-Román, R. M., Guillot-Ferriols, M., Roig-Pérez, L., Lanceros-Mendez, S., Gallego-Ferrer, G., & Gómez Ribelles, J. L. (2019). Freeze-extraction microporous electroactive supports for cell culture. European Polymer Journal, 119, 531-540. doi:10.1016/j.eurpolymj.2019.07.011Camacho, N. P., West, P., Torzilli, P. A., & Mendelsohn, R. (2000). FTIR microscopic imaging of collagen and proteoglycan in bovine cartilage. Biopolymers, 62(1), 1-8. doi:10.1002/1097-0282(2001)62:13.0.co;2-oRibeiro, C., Panadero, J. A., Sencadas, V., Lanceros-Méndez, S., Tamaño, M. N., Moratal, D., … Gómez Ribelles, J. L. (2012). Fibronectin adsorption and cell response on electroactive poly(vinylidene fluoride) films. Biomedical Materials, 7(3), 035004. doi:10.1088/1748-6041/7/3/035004Ribeiro, C., Pärssinen, J., Sencadas, V., Correia, V., Miettinen, S., Hytönen, V. P., & Lanceros‐Méndez, S. (2014). Dynamic piezoelectric stimulation enhances osteogenic differentiation of human adipose stem cells. Journal of Biomedical Materials Research Part A, 103(6), 2172-2175. doi:10.1002/jbm.a.35368Sobreiro-Almeida, R., Tamaño-Machiavello, M., Carvalho, E., Cordón, L., Doria, S., Senent, L., … Sempere, A. (2017). Human Mesenchymal Stem Cells Growth and Osteogenic Differentiation on Piezoelectric Poly(vinylidene fluoride) Microsphere Substrates. International Journal of Molecular Sciences, 18(11), 2391. doi:10.3390/ijms18112391Moise, S., Céspedes, E., Soukup, D., Byrne, J. M., El Haj, A. J., & Telling, N. D. (2017). The cellular magnetic response and biocompatibility of biogenic zinc- and cobalt-doped magnetite nanoparticles. Scientific Reports, 7(1). doi:10.1038/srep3992

Dual cholinergic signals regulate daily migration of hematopoietic stem cells and leukocytes.

Hematopoietic stem and progenitor cells (HSPCs) and leukocytes circulate between the bone marrow (BM) and peripheral blood following circadian oscillations. Autonomic sympathetic noradrenergic signals have been shown to regulate HSPC and leukocyte trafficking, but the role of the cholinergic branch has remained unexplored. We have investigated the role of the cholinergic nervous system in the regulation of day/night traffic of HSPCs and leukocytes in mice. We show here that the autonomic cholinergic nervous system (including parasympathetic and sympathetic) dually regulates daily migration of HSPCs and leukocytes. At night, central parasympathetic cholinergic signals dampen sympathetic noradrenergic tone and decrease BM egress of HSPCs and leukocytes. However, during the daytime, derepressed sympathetic noradrenergic activity causes predominant BM egress of HSPCs and leukocytes via β3-adrenergic receptor. This egress is locally supported by light-triggered sympathetic cholinergic activity, which inhibits BM vascular cell adhesion and homing. In summary, central (parasympathetic) and local (sympathetic) cholinergic signals regulate day/night oscillations of circulating HSPCs and leukocytes. This study shows how both branches of the autonomic nervous system cooperate to orchestrate daily traffic of HSPCs and leukocytes

Remodeling of Bone Marrow Hematopoietic Stem Cell Niches Promotes Myeloid Cell Expansion during Premature or Physiological Aging.

Hematopoietic stem cells (HSCs) residing in the bone marrow (BM) accumulate during aging but are functionally impaired. However, the role of HSC-intrinsic and -extrinsic aging mechanisms remains debated. Megakaryocytes promote quiescence of neighboring HSCs. Nonetheless, whether megakaryocyte-HSC interactions change during pathological/natural aging is unclear. Premature aging in Hutchinson-Gilford progeria syndrome recapitulates physiological aging features, but whether these arise from altered stem or niche cells is unknown. Here, we show that the BM microenvironment promotes myelopoiesis in premature/physiological aging. During physiological aging, HSC-supporting niches decrease near bone but expand further from bone. Increased BM noradrenergic innervation promotes β2-adrenergic-receptor(AR)-interleukin-6-dependent megakaryopoiesis. Reduced β3-AR-Nos1 activity correlates with decreased endosteal niches and megakaryocyte apposition to sinusoids. However, chronic treatment of progeroid mice with β3-AR agonist decreases premature myeloid and HSC expansion and restores the proximal association of HSCs to megakaryocytes. Therefore, normal/premature aging of BM niches promotes myeloid expansion and can be improved by targeting the microenvironment.We thank A.R. Green for advice and support; M. García-Fernández, C. Fielding, C. Kapeni, X. Langa, and other current and former members of the S.M.-F group for help and discussions; A. Barettino and A. Macías (CNIC), D. Pask, T. Hamilton, the Central Biomedical Services and Cambridge NIHR BRC Cell Phenotyping Hub for technical assistance; H. Jolin and A.N.J. McZenzie (MRC Laboratory of Molecular Biology, Cambridge, UK) for help with milliplex analyses. Y.-H.O. received fellowships from Alborada Scholarship (University of Cambridge), Trinity-Henry Barlow Scholarship (University of Cambridge) and R.O.C. Government Scholarship to Study Abroad (GSSA) A.G.G. received fellowships from Ramón Areces and LaCaixa Foundations. C.K. was supported by Marie Curie Career Integration grant H2020-MSCA-IF-2015-70841. S.M.F., by Red TerCel (ISCIII-Spanish Cell Therapy Network). VA is supported by grants from the Spanish Ministerio de Economía, Industria y Competitividad (MEIC) with cofunding from the Fondo Europeo de Desarrollo Regional (FEDER, “Una manera de hacer Europa”) (SAF2016-79490-R), the Instituto de Salud Carlos III (AC16/00091), the Fundació Marató TV3 (122/C/2015), and the Progeria Research Foundation (Established Investigator Award 2014–52). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia, Innovación y Universidades (MCNU) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505). This work was supported by core support grants from the Wellcome Trust and the MRC to the Cambridge Stem Cell Institute, the Spanish Ministry of Economy and Competitiveness (SAF-2011-30308), Ramón y Cajal Program grant RYC-2009-04703, ConSEPOC-Comunidad de Madrid S2010/BMD-2542, National 427 Health Service Blood and Transplant (United Kingdom), European Union’s Horizon 428 2020 research (ERC-2014-CoG-64765 and Marie Curie Career Integration grant FP7- 429 PEOPLE-2011-RG-294096) and a Programme Foundation Award from Cancer Research 430 UK to S.M.-F., who was also supported in part by an International Early Career Scientist 431 grant of the Howard Hughes Medical Institute

Additional value of screening for minor genes and copy number variants in hypertrophic cardiomyopathy

Introduction: Hypertrophic cardiomyopathy (HCM) is the most prevalent inherited heart disease. Next-generation sequencing (NGS) is the preferred genetic test, but the diagnostic value of screening for minor and candidate genes, and the role of copy number variants (CNVs) deserves further evaluation. Methods: Three hundred and eighty-seven consecutive unrelated patients with HCM were screened for genetic variants in the 5 most frequent genes (MYBPC3, MYH7, TNNT2, TNNI3 and TPM1) using Sanger sequencing (N = 84) or NGS (N = 303). In the NGS cohort we analyzed 20 additional minor or candidate genes, and applied a proprietary bioinformatics algorithm for detecting CNVs. Additionally, the rate and classification of TTN variants in HCM were compared with 427 patients without structural heart disease. Results: The percentage of patients with pathogenic/likely pathogenic (P/LP) variants in the main genes was 33.3%, without significant differences between the Sanger sequencing and NGS cohorts. The screening for 20 additional genes revealed LP variants in ACTC1, MYL2, MYL3, TNNC1, GLA and PRKAG2 in 12 patients. This approach resulted in more inconclusive tests (36.0% vs. 9.6%, p<0.001), mostly due to variants of unknown significance (VUS) in TTN. The detection rate of rare variants in TTN was not significantly different to that found in the group of patients without structural heart disease. In the NGS cohort, 4 patients (1.3%) had pathogenic CNVs: 2 deletions in MYBPC3 and 2 deletions involving the complete coding region of PLN. Conclusions: A small percentage of HCM cases without point mutations in the 5 main genes are explained by P/LP variants in minor or candidate genes and CNVs. Screening for variants in TTN in HCM patients drastically increases the number of inconclusive tests, and shows a rate of VUS that is similar to patients without structural heart disease, suggesting that this gene should not be analyzed for clinical purposes in HCM

Different niches for stem cells carrying the same oncogenic driver affect pathogenesis and therapy response in myeloproliferative neoplasms

Aging facilitates the expansion of hematopoietic stem cells (HSCs) carrying clonal hematopoiesis-related somatic mutations and the development of myeloid malignancies, such as myeloproliferative neoplasms (MPNs). While cooperating mutations can cause transformation, it is unclear whether distinct bone marrow (BM) HSC-niches can influence the growth and therapy response of HSCs carrying the same oncogenic driver. Here we found different BM niches for HSCs in MPN subtypes. JAK-STAT signaling differentially regulates CDC42-dependent HSC polarity, niche interaction and mutant cell expansion. Asymmetric HSC distribution causes differential BM niche remodeling: sinusoidal dilation in polycythemia vera and endosteal niche expansion in essential thrombocythemia. MPN development accelerates in a prematurely aged BM microenvironment, suggesting that the specialized niche can modulate mutant cell expansion. Finally, dissimilar HSC-niche interactions underpin variable clinical response to JAK inhibitor. Therefore, HSC-niche interactions influence the expansion rate and therapy response of cells carrying the same clonal hematopoiesis oncogenic driver